Tramadol is an analgesic drug that binds to specific opioid receptors. It may contribute to the inhibition of neuronal re-uptake of noradrenaline. Catalase is a key enzyme for degrading H2O2 in cells and has various isoforms with different structures and kinetics properties. In this research, the effect of tramadol on the activity of catalase of Pseudomonas and mouse liver was investigated and compared. Tramadol could inhibit Pseudomonas catalase with mixed inhibition while inhibiting mouse liver catalase in a non-competitive manner. The Ki and IC50 values were determined as 0.45 and 1.5 mM for Pseudomonas and 2.8 and 2.5 mM for mouse liver, respectively. SDS-PAGE of partially purified catalases determined different molecular weights for Pseudomonas (Mw 54 kDa) and mouse liver (61 kDa).