Staphylococcus aureus (S. aureus) is one of the most common pathogens for nosocomial and community infections, which is closely related to the occurrence of pyogenic and toxic diseases in human beings. In the current study, a lab-built microchip capillary electrophoresis (microchip CE) system was employed for the rapid determination of S. aureus, while a simple-to-use space domain internal standard (SDIS) method was carried out for the reliable quantitative analysis. The precision, accuracy, and reliability of SDIS were investigated in detail. Noted that these properties could be elevated in SDIS compared with traditional IS method. Remarkably, the PCR products of S. aureusnuc gene could be identified and quantitated within 80 s. The theoretical detection limit could achieve a value of 0.066 ng/μL, determined by the using SDIS method. The current work may provide a promising detection strategy for the high-speed and highly efficient analysis of pathogens in the fields of food safety and clinical diagnosis.