In this short communication, we report simultaneous measurement of CO and OH in flames using a single femtosecond (fs)-duration laser source. Two-photon excitation of CO A1Π ←←X1Ʃ+(3,0), (4,0) and (5,0) bands and single-photon excitation of OH A2Ʃ+←X2Π (1,0) band are achieved simultaneously using one fs-duration laser pulse near 283 nm. Subsequently, crosstalk-free, laser-induced fluorescence (LIF) emissions are detected from the v’=3, 4 and 5 upper vibrational levels of CO in the 200–230 nm region, and (1,1) and (0,0) bands of OH near the 310-nm region. A detailed spectroscopic investigation of CO in a gas cell is followed by simultaneous detection of CO and OH in C2H4/air flames. The measured CO and OH profiles as a function of flame equivalence ratio agree well with equilibrium calculations. The present study extends the applicability of broadband fs-LIF imaging of OH in flames, and reveal an alternate method to obtain heat release rate in turbulent flames via simultaneous OH and CO measurements.