A bioactive packaging material based on chitosan (CS) incorporated with benzyl isothiocyanate (BITC) and α−cyclodextrin (α−CD) was fabricated to evaluate its preservative effects on fresh beef stored at 4 °C for 12 d according to the quality analysis. The Fourier-transform infrared (FTIR) spectrum revealed that the major structural moiety of BITC was embedded in the cavity of α−CD, except for the thiocyanate group. FTIR and X-ray diffraction analysis further verified that intermolecular interactions were formed between the BITC−α−CD and CS film matrix. The addition of BITC−α−CD decreased the UV light transmittance of pure CS film to lower than 63% but still had enough transparency for observing packaged items. The CS−based composite film displayed a sustainable antibacterial capacity and an enhanced antioxidant activity. Moreover, the total viable counts, total volatile base nitrogen, pH, thiobarbituric acid–reactive substances, and sensory evaluation of the raw beef treated with the CS−based composite film were 6.31 log colony-forming unit (CFU)/g, 19.60 mg/100 g, 6.84, 0.26 mg/kg, and 6.5 at 12 days, respectively, indicating the favorable protective efficacy on beef. These results suggested that the fabricated CS−based composite film has the application potential to be developed as a bioactive food packaging material, especially for beef preservation.