Engineering Metal–Organic Framework Nanosheets as High-Performance Peroxidase Mimics for Visual Detection of African Swine Fever Virus

African swine fever virus (ASFV) poses a severe threat to the global swine industry, demanding rapid on-site diagnostics due to the lack of an effective vaccine. Existing laboratory-based methods, such as PCR and ELISA, are limited by cost, complexity, and infrastructure requirements, hindering their use in resource-limited settings. Colorimetric assays offer a promising alternative for instrument-free, rapid detection, but conventional reliance on fragile and expensive natural enzymes such as horseradish peroxidase (HRP) is problematic. This research addresses this need by developing a highly sensitive colorimetric sensor for ASFV based on a two-dimensional (2D) MoSe2@Fe-MOF nanozyme. This composite nanozyme leverages a synergistic effect: Fe-MOF provides abundant peroxidase-like (POD-like) active sites, while integrated MoSe2, a p-type semiconductor, enhances charge transfer at the heterojunction. This synergy boosts the catalytic oxidation of the chromogenic substrate TMB, resulting in a vibrant blue color signal (the “ON” state). To achieve specific ASFV detection, the nanozyme surface is functionalized with antibodies targeting the p72 major capsid protein. Upon capturing ASFV, the resulting immuno-complex generates significant steric hindrance, physically blocking catalytic sites and impeding substrate access. This inhibition significantly reduces TMB oxidation, leading to a decrease in color development and absorbance (the “OFF” state). This efficient signal modulation enables the sensor to achieve a remarkably low limit of detection of 0.22 TCID50/mL in just 15 min, offering a rapid, cost-effective, and reliable solution for on-site ASFV diagnostics.