Infectious diseases caused byStreptococcus iniaelead to massive death of fish, compose a serious threat to the global aquaculture industry, and constitute a risk to humans who deal with raw fish. In order to realize the early diagnosis ofS. iniae, and control the outbreak and spread of disease, it is of great significance to establish fast, sensitive, and convenient detection methods forS. iniae. In the present study, two methods of real-time MIRA (multienzyme isothermal rapid amplification, MIRA) and MIRA-LFD (combining MIRA with lateral flow dipsticks (LFD)) for thesimAgene ofS. iniaewere established, which could complete amplification at a constant temperature of 42 °C within 20 min. Real-time MIRA and MIRA-LFD assays showed high sensitivity (97 fg/μL or 7.6 × 102CFU/mL), which were consistent with the sensitivity of real-time PCR and 10 times higher than that of PCR with strong specificity, repeatability simplicity, and rapidity forS. iniaeoriginating fromTrachinotus ovatus. In summary, real-time MIRA and MIRA-LFD provide effective ways for early diagnosis ofS. iniaein aquaculture, especially for units in poor conditions.