Electrochemical behavior of myoglobin (Mb) incorporated in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Mb-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks for Mb Fe(III)/Fe(II) redox couple at about -0.27 (vs. SCE) in pH 5.5 buffers. The electron exchange of Mb with PG electrodes was greatly enhanced in PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting the data of square wave voltammetry (SWV) with non-linear regression analysis. The formal potential of Mb-PAM films shifted linearly with pH with a slope of -0.52 V, showing the electron transfer was accompanied by a single-proton transportation. Positions of Soret absorbance band of Mb-PAM films suggest that Mb maintains its secondary structure similar to its native state in the films in the medium pH range. Oxygen, trichloroacetic acid (TCA) and nitrite were catalytically reduced by Mb-PAM film electrodes with significant lowering of overpotential. Potential application of Mb-PAM films as biosensors to monitor some substrates was proposed.