In purpose of valid Streptococcus iniae detection, we established a colorimetric biosensor using gold nanoparticles (AuNPs) labeled with dual functional probes and along with loop-mediated isothermal amplification (LAMP) assay (LAMP-AuNPs). Based on the characteristics of self-aggregation and bio-conjugation with ligands, AuNPs were chosen for observable color change in tandem with LAMP amplification method to reach high sensitivity and easy operation. Meanwhile, the improvement of dual probes that could fully utilize the LAMP product gave the biosensor a stable result exhibition. LAMP-AuNPs targeting gene ftsB, one of the ATP transporter-related genes, turned out favorable specificity in cross reaction among other fish pathogens. The detect limit of 10(2) CFU revealed a better sensitivity compared with polymerase chain reaction (PCR) method and AuNPs lateral flow test strip (LFTS). It was also proved to be effective by zebrafish infection model trials with less than 2-h time consumption and nearly no devices which make it a convenient biosensor for point-to-care S. iniae detection.