Nanozymes have become a frontier of research recently and possess broad application prospects in the field of analytical sensing. Herein, a Fe–Mn bimetallic nanozyme (FeMnzyme) was proposed for colorimetric analysis of ascorbic acid (AA). The FeMnzyme, with high specific surface area, consisted of Fe–Mn mixed oxide amorphous core and α-FeOOH nanorod shell characterized by scanning electron microscope (SEM) and Powder X-ray diffraction (XRD). It exhibits typical oxidase-like properties to catalyze oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to generate visible blue oxidized TMB (oxTMB) without exogenous H2O2. Due to the reducibility of AA to oxTMB, the FeMnzyme/oxTMB system can be employed as a promising colorimetric sensor for quantitative analysis of AA. The method has a good linear concentration range of 8 μmol L−1 to 56 μmol L−1, and the detection limit is as low as 0.88 μmol L−1 (3σ). Finally, the proposed biosensor has been favorably put into use to determine AA content in kiwi fruit, indicating its reliability for practical application.