After working as a mechanic for various Manchester machine manufacturers, Whitworth went to London in 1825 and at Maudslay & Company devised a scraping technique for making a true plane surface. Returning to Manchester in 1833, he opened his own toolmaking business. Between 1840 and 1850 he produced an original measuring machine and a system of accurate dimensional standards or master gauges to go with it. Even the common screw was not overlooked. In 1841 Whitworth's standard screw threads were adopted by the Woolwich Arsenal.

By 1851 Whitworth's machine tools had become internationally known for their accuracy and quality. He had exhibited his screw cutting lathes, his planing, drilling, slotting, and shaping machines, and his millionth-part measuring machine. By 1866 his factory employed 700 men and was equipped with 600 machine tools. He also did pioneering work in ordnance, inventing a method for casting ductile steel to replace hard steel, which is subject to fracture.

Whitworth helped found the chair of engineering and laboratories at Owens College, Manchester. In 1868 he established the Whitworth scholarships, setting aside an annual sum of £3,000 for the purpose. In 1869 he was created a baronet.