Dufferin runs from just north of the city's waterfront at the intersection of Queen's Quay and Fleet Street in the south, to the city's northern border with Vaughan, where it becomes York Regional Road 53.

Along its route, Dufferin Street passes through several diverse neighborhoods, including Liberty Village, Parkdale, Brockton Village, Bloordale Village, Corso Italia, Fairbank, and Glen Park. It also intersects with a number of major east-west streets, including Queen Street West, College Street, Bloor Street West, St. Clair Avenue West, Eglinton Avenue West, and Lawrence Avenue West.

Dufferin Street is well-served by public transit, with several TTC (Toronto Transit Commission) bus routes running along its length, as well as the 29 Dufferin and 511 Bathurst streetcar lines. The Dufferin subway station, located at the intersection of Dufferin Street and Bloor Street West, provides access to Line 2 (Bloor-Danforth) of the TTC subway system.