To investigate the flexural behavior of steel-concrete composite beams strengthened with prestressed carbon fiber-reinforced polymer (CFRP) plates, a total of five large scale steel-concrete composite beams including one control unstrengthened beam and four strengthened beams were prepared and tested with static load in a simply supported four-point bending setup. Welded I-beam was used for the structural steel section of all specimens. An innovative fabricated anchorage system was developed to prestress the CFRP plates attached to the bottom flange of the steel beams. The connection between the CFRP plates and the steel beams both depended on flat-plate anchors and epoxy resin adhesive. The strengthening effects of the method using CFRP plates of different thicknesses and different prestressing forces were investigated. Experimental results showed that the strain distribution along the cross-section conformed to plane-section assumption during load application. The specimen strengthened with thicker CFRP plate exhibited a higher ultimate bearing capacity. The yield bearing capacity increased and the mid-span deflection slightly decreased with increase of prestressing force. The CFRP plates failed due to rupture or debonding under ultimate condition and a great portion of tensile strength was employed (up to 80% on average). The fabricated anchorage system can provide strong anchoring force and is proved to be feasible and practical. The prestressed CFRP plate for strengthening steel-concrete beams improves the flexural behavior and is an efficient technique in bridge strengthening and rehabilitation.