Aims: Staphylococcus aureus (Staph. aureus) produces a wide variety of staphylococcal enterotoxins (SEs) and staphylococcal enterotoxin-like (SEl) proteins, which are the most causative agents of staphylococcal food poisoning. In contrast to classical SEs (SEA to SEE), the relationship between the novel SEs/SEls (SEG to SElX) and staphylococcal food poisoning is not elucidated. This study is aimed to establish a system to detect staphylococcal enterotoxin-like protein I (SElI) for analysis of staphylococcal food poisoning. Methods and Results: SElI was characterized in a Staph. aureus clinical isolate associated with food poisoning; there was an amino acid substitution Thr145Ala compared to previously identified SEI from Staph. aureus 04-02981. Subsequently, SElI was expressed, purified, and the poly-and monoclonal antibodies against it were prepared. Using these antibodies, a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that specifically detected and measured SElI secretion from the Staph. aureus clinical isolate in LB medium, milk and bloodstream was developed. Conclusions: The ELISA system has been successfully applied for analysing SElI secretion in vivo and in vitro. Significance and Impact of the Study: The highly sensitive ELISA should make it attractive for quantifying SElI in food hygiene supervision and clinical diagnosis in near future.