Excessive use of antibiotics in aquaculture severely endangers human health and ecosystems, which has raised significant concerns in recent years. However, conventional laboratory-based approaches regularly required time or skilled manpower. Herein, we propose a point-of-care-testing (POCT) biosensor detection device for the simultaneous determination of multiantibiotics without complex equipment or professional operators. A laser-printed paper-based microfluidic chip loaded with multicolor fluorescence nanoprobes (mCD-μPAD) was developed to rapidly detect sulfamethazine (SMZ), oxytetracycline (OTC), and chloramphenicol (CAP) on-site. These “fluorescence off” detection probes composed of carbon dots (CDs) conjugated with aptamers (donor) and MoS2 nanosheets (acceptor) (CD-apt-MoS2) were based on Förster resonance energy transfer. Upon the addition of target antibiotics, the significantly recovered fluorescence signal on the μPAD can be sensitively perceived by employing a 3D-printed portable detection box through a smartphone. Under optimal conditions, this μPAD allowed for a rapid response of 15 min toward SMZ, OTC, and CAP with considerable sensitivities of 0.47, 0.48, and 0.34 ng/mL, respectively. In shrimp samples, the recoveries were 95.2–101.2, 96.4–105, and 96.7–106.1% with RSD below 6%. This paper-based sensor opens an avenue for on-site, high-throughput, and rapid detection methods and can be widely used in POCT in food safety.

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