We demonstrate a femtosecond two-photon laser-induced fluorescence (fs-TPLIF) technique for sensitive CO detection, using a 230 nm pulse of 9 µJ and 45 fs. The advantages of fs-TPLIF in excitation of molecular species were analyzed. Spectra of CO fs-TPLIF were recorded in stable laminar flames spatially resolved across the flame front. A hot band (1, n) together with the conventional band (0, n) of the B→A transitions were observed in the burned zone and attributed to the broadband nature of the fs excitation. The CO fs-TPLIF signal recorded across the focal point of the excitation beam shows a relatively flat intensity distribution despite of the steep laser intensity variation, which is beneficial for CO imaging in contrast to nanosecond and picosecond TPLIF. This phenomenon can be explained by photoionization, which over the short pulse duration dominates the population depletion of the excited B state due to the high peak power, but only contributes in total a negligible X state depletion due to the low pulse energy. Single-shot CO fs-TPLIF images in methane/air flames were recorded by imaging the broadband fluorescence. The results indicate that fs-TPLIF is a promising tool for CO imaging in flames.