Conventional plating methods for enumerating Staphylococcus aureus are time-consuming and labor-intensive. Rapid methods for specific quantification are desirable. Fluorescence in situ hybridization with filter-cultivation (FISHFC) method has been already developed to enumerate viable specific microorganisms such as Listeria monocytogenes , Clostridium perfringens and Vibrio parahaemolyticus . The purpose of this study was to develop and estimate FISHFC method for S. aureus quantitative detection in food samples. Alexa Fluor^® 546-labeled oligonucleotide probe, STA68, was newly designed from the 16S rRNA gene sequences of S. aureus . STA68-conferred fluorescence was observed for S. aureus but not for any other organisms, suggesting that STA68 probe is highly specific for S. aureus . Results were achievable within 12 hours by FISHFC method, containing with 10 hours cultivation, as compared to more than 3 days required for confirmation of S. aureus by conventional plating methods. When inoculated to BPW and food samples, the numbers of viable counts determined by FISHFC method were not significantly different to those obtained by the conventional plating method ( p >0.05). This result suggests that FISHFC method was preferable for the specific, rapid and accurate quantification of viable S. aureus in food.