Whitworth was born in Stockport and at a young age developed an interest in machinery. He worked as a mechanic in Manchester and then in London for Henry Maudslay, Holtzapfel and John Clement. At Clement's workshop he helped with the manufacture of Charles Babbage's calculating machine. He returned to Openshaw, near Manchester, in 1833 to start his own business manufacturing lathes and other machine tools, which were renowned for their high standard of workmanship.
He invented a method of producing accurate flat surfacess using three trial surfaces, in 1830. This used engineer's blue and three trial surfaces. This led to an explosion of development of precision instruments using his flat surfaces as a basis for further construction of precise shapes.
His next innovation, in 1840, was a measuring technique called "end measurements" that used a precision flat plane and measuring screw, both of his own invention. The system, with an accuracy of one millionth of an inch, was demonstrated at the Great Exhibition of 1851.
In 1841 Whitworth devised the first standardised system for screw threads. Its adoption by the railway companies, who until then had all used different screw threads, led to its widespread acceptance. It later became a British Standard, "British Standard Whitworth", abbreviated to BSW and governed by BS 84:1956.
Whitworth was commissioned by the War Department of the British government to design a replacement for the Enfield rifle, whose shortcomings had been revelaed during the recent Crimean War. The Whitworth rifle had a smaller bore (0.45 inch) which was hexagonal, a longer bullet and tighter rifling than the Enfield, and its performance during tests in 1859 was superior to the Enfield in every way. The test was reported in The Times on April 23 as a great success. However, the new bore design was found to be prone to fouling, so it was rejected by the British government, only to be adopted by the French Army. Some of the rifles found their way to the Confederate states in the American Civil War, where they were called "Whitworth Sharpshooters".
Queen Victoria opened the first meeting of the British Rifle Association at Wimbledon in 1860 by firing a Whitworth Sharpshooter from a mechanical rest. The rifle scored a bull's eye at a range of 400 yards.
Whitworth also designed a large rifled cannon with a 2.75 inch bore, a 12 pound 11 ounce projectile and a range of about six miles. The spirally-grooved projectile was patented in 1855. This was also rejected by the British army, but was also used in the American Civil War.
While trying to increase the bursting strength of his gun barrels, Whitworth patented a process called "fluid-compressed steel" for casting steel under pressure, and built a new steel works near Manchester. Some of his castings were shown at the Great Exhibition in Paris ca. 1883.
Whitworth received many awards for the excellence of his designs, and was financially very successful. In 1850, then a Fellow of the Royal Society and President of the Institution of Mechanical Engineers, he built a house called The Firs, in Fallowfield, south of Manchester. In 1854 he bought Stancliffe Hall in Darley Dale, Derbyshire. In 1872 he moved there with his second wife.
A strong believer in the value of techical education, Whitworth backed the new Mechanics Institute in Manchester, and helped found the Manchester School of Design. In 1868 he founded a scholarship for the advancement of mechanical engineering.
Whitworth died at Monte Carlo, where he had travelled in the hope of improving his health. He was buried at the church of Darley (or Darley Dale) St Helen in Derbyshire. A detailed obituary was published in the American magazine The Manufacturer and Builder (Volume 19, Issue 6, June 1887). He directed his trustees to spend his fortune on philanthropic projects, which they still do to this day.