The interaction behavior of bovine serum albumin (BSA), lysozyme (LYS), myoglobin (MB), and catalase (CAT) with luminol, respectively, was first studied by chemiluminescence (CL) using flow injection (FI) technique based on the fact that the studied proteins can enhance the CL intensity of luminol. A FI-CL model of protein-luminol interaction, lg[( 0 − )/ ] = 1/ lg[ ]+1/ lg + 2lg , was constructed, and the interaction parameters of BSA, LYS, MB, and CAT with luminol were determined accordingly. The binding constants are in the descending order of CAT > MB > LYS > BSA at the level of 10 5 to 10 7 L mol −1 , and the number of binding sites of luminol to BSA or LYS is around 2 and to MB or CAT is around 1. The results of thermodynamic parameters (Δ , Δ , and Δ ) showed that the binding processes of luminol to the four proteins are spontaneous mainly through the hydrophobic force.