In this work, graphene oxide (GO) sheets were assembled within the porous calcium alginate (CA) films, and applied to fabricate electroactive biosensor by incorporating with myoglobin (Mb). The porous Mb-GO-CA films were fabricated and characterized by scanning electron microscopy (SEM), UV-Vis adsorption, and electrochemistry techniques. UV-Vis spectrum demonstrated that Mb retained its secondary structure in the porous Mb-GO-CA films at medium pH. Cyclic voltammetry of the porous Mb-GO-CA films showed a pair of well-defined and nearly reversible peaks for MbFe(III)/Fe(II) redox couple at -0.350 V versus saturated calomel electrode in pH 7.0 buffers. The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k (s)) and formal potential (E (omicron)') were estimated by fitting the data of square-wave voltammetry with nonlinear regression analysis. The porous Mb-GO-CA films exhibited excellent electrocatalytic reduction to sodium nitrite, oxygen, and hydrogen peroxide, and showed good reproducibility and stability, which may provide a promising platform for the fabrication of the third-generation biosensor.