It has been reported in many studies that piperine (PIP) has multiple activity properties, the most prominent of which is antioxidant activity. This work reports the binding behavior and antioxidant activity of the spice extract piperine towards myoglobin (Mb) using spectroscopic and fluorescence spectra analysis, and computational approaches. Antioxidant activity studies have shown that the antioxidant effect of the Mb-PIP complex system depends on the concentration of PIP added. An appropriate concentration of PIP can successfully prevent the release of free iron from Mb. The fluorescence results indicated that the binding of PIP to Mb was via static quenching. After binding to PIP, the α-helix content of Mb decreased by about 5%. Synchronous fluorescence results indicate that PIP is closer to Trp, and MD simulations also demonstrate that PIP enters the hydrophobic cavity of Mb and binds stably. This explains the structural changes in proteins that lead to changes in antioxidant properties. The results of this study provide a reference for the quality control of additives of plant origin in the processing and storage of meat and meat products.