Fluorescence nano-sensors have recently emerged as a cost-effective, convenient, and time-saving tool for high-throughput detection of antibiotics in wastewater. However, their application is primarily limited to tetracycline and penicillin due to a lack of understanding of key influencing factors. Herein, we propose a convenient strategy to expand their application range by increasing the colors of fluorescence nano-sensors. The newly three-colored nano-sensors were prepared to detect erythromycin and metronidazole, which has rarely been reported before. For in-situ detection, a smartphone app-based detection method was developed, and the detection method was optimized to obtain the widest detection limit. The fluorescence nano-sensors demonstrated satisfactory recovery rates with promising accuracy in various types of wastewaters. Moreover, a mechanism investigation comprising experimental analysis and theoretical simulation was developed to elucidate their detection mechanisms and provide ideas for improving their performance. Consequently, several improvements were proposed further to enhance the analysis selectivity, sensitivity, and accuracy. This work presents a new perspective on broadening the application of fluorescence nano-sensors and provides a novel route to analyze the detection mechanism, enabling rapid, high-throughput and in-situ detection of antibiotics.