Nano-biosynthesis for gold nanoparticle (AuNP) using bacteria can produce the tailored functional AuNP because of the different bioactive molecules coating on the AuNP derived from different bacteria. However, the biosynthesis for the biocompatible AuNP from the harmful bacteria is still challenging, and the extensive application of the bacteria-derived AuNP in tissue repair is lacking. In this study, without other auxiliary chemical molecules, the gradient centrifugation was used to successfully remove the toxic part of the pristine AuNPs biosynthesized from Staphylococcus aureus (S. aureus). The purified S. aureus-derived AuNPs were proved to be beneficial for the muscle cells’ viability and could even protect the cells against the cardiotoxin damages. Furthermore, the S. aureus-derived AuNPs were assembled into an elastic scaffold to form the AuNPs-incorporated cardiac patch. The in vivo study in rat myocardial infarction (MI) models demonstrated that these S. aureus-derived AuNPs could be taken as a functional factor in the cardiac patch to promote MI repair, through decreasing the infarct area and improving the cardiac function of the infarct heart. This study provides a functional S. aureus-derived AuNP with tissue repair potential, which can be extensively applied in muscle tissue engineering.