<p>A four-channel fluorescent sensor array was developed for the detection of rifamycin antibiotics (RFs), which is a broad-spectrum antibiotic. This sensor array was composed of two synthetic carbon dots (B1 and S1) and two additional sensors (B2 and S2), which were derived from the mixture of B1 and S1 with chromium ions (Cr³⁺), respectively. The distinct interactions between individual sensors and specific RFs produced varying fluorescence quenching responses. These differential optical signals, when analyzed through a pattern recognition algorithm, enabled the identification of five distinct RFs (rifampicin, rifapentine, rifabutin, rifaximin, and rifamycin S) in both individual and binary mixture samples, maintaining robust differentiation capability. Additionally, the sensor array revealed the capability to quantitatively assess rifampicin and rifapentine with limits of detection of 45 nM and 87 nM, respectively (<i>n</i> = 6). Notably, the good interference immunity of the sensor array enabled accurate discrimination of five RFs and quantification of rifampicin and rifapentine in lake water and bovine serum albumin. This study can identify five RFs and quantitatively determine rifampicin and rifapentine with excellent selectivity, reproducibility, and practicality.</p> Graphical abstract <p></p>