Cholesterol, the primary component of cell membranes, plays a critical role in wound healing and antibacterial defense. Recently, various reactive oxygen species-mediated strategies have been developed for disinfecting drug-resistant microorganisms. However, development of low-cost cholesterol consumption-based enzyme-mimicking platforms with high antibacterial efficacy remains a significant challenge. Herein, we report the synthesis of a laminated Au-TiO2/C nanozyme, which shows excellent peroxidase-like activity in antibacterial applications. The resultant Au-TiO2/C nanozyme can effectively convert H2O2 into •OH, demonstrating efficient bactericidal activity against Escherichia coli and Staphylococcus aureus. Moreover, based on the prominent peroxidase-like activity of Au-TiO2/C and the cascade reaction of cholesterol oxidase, a sensitive and selective cholesterol colorimetric biosensor was developed. The biosensor exhibits high sensing performance toward cholesterol detection, with a linear detection range from 25 to 300 μM and a detection limit of 13.22 μM. It can also rapidly and accurately detect cholesterol in real serum samples. These findings highlight the dual functionality of the Au-TiO2/C nanozyme as both an effective bactericide and a cholesterol sensor. Hence, the study could contribute great potential to the fast detection and sterilization of multilayer Au-TiO2/C nanozymes in the biomedical field.