Carbon monoxide (CO) and hydroxyl (OH) are the two key intermediate species formed during chemical reactions inside gas turbine combustors. Spatiotemporal information and a detailed understanding of CO formation in the reaction zone are important during the combustion processes as a major part of the heat release is obtained from the oxidation of CO to CO2. Turbulent flame structures and reaction zone in different flame conditions can also be visualized through the spatial distribution profiles of OH. In the current study, both these species are excited simultaneously using a single ultrashort, broad spectral bandwidth of approximately 100-femtosecond (fs) duration laser pulse at λ = 283.8 nm. Subsequent fluorescence signals are separated through spectral filters of appropriate bandwidth and imaged using two cameras. This present study was performed in a McKenna flat-flame burner with ethylene/air as a pilot flame and non-premixed turbulent ethylene jet at the center. The partial spectral overlap of CO–X (4,0) and OH A–X (1,0) transitions are utilized for simultaneous excitation, thereby characterize the overall flame structure (via OH) and regions of oxidation reactions (via CO) in a range of flame conditions. Besides, CO and OH profiles follow the trends obtained from model predictions for a range of equivalence ratios in ethylene/air flames stabilized over the Hencken calibration burner. These results are used for obtaining quantitative calibrations of CO and OH signals. Overall, the present study extends the applicability of a single, broadband fs laser pulse for simultaneous imaging of multiple chemical species in flame.