β-Lactam antibiotics such as penicillins and cephalosporins are extensively used for human infection therapy. Consistent unintended exposure to these antibiotics via food and water is known to promote antibiotic-resistant bacterial pathogenesis with high morbidity and mortality in humans. An optical enzymatic biosensor for rapid and point-of-use detection of these antibiotics in food and water has been developed and tested. Enzymatic hydrolysis of β-lactams, on the electroactive polyaniline nanofibers, altered the polymeric backbone of the nanofibers, from emeraldine base form to emeraldine salt, which was measured as an increase in evanescent wave absorbance at 435 nm. The sensors were calibrated by spiking antibiotic-free milk with ceftazidime (as a model β-lactam analyte) in a linear range of 0.36–3600 nM (R2 = 0.98). The calibration was further validated for packaged milk, local cow milk, and buffalo milk. A similar calibration was devised for chicken meat samples in a linear range of 9–1800 nM (R2 = 0.982) and tap water in a linear range of 0.18–180 nM (R2 = 0.99). Interestingly, it was possible to use the same calibration for the determination of other β-lactam antibiotics (ampicillin, amoxicillin, and cefotaxime), which reflects the usefulness of the sensor for wide-scale deployment. The sensor performance was validated with a wastewater sample, from a wastewater treatment plant (WWTP), qualitatively analyzed by high-resolution liquid chromatography coupled with mass spectroscopy for detection of β-lactams. The sensor scheme developed and tested is of grassroot relevance as a quick solution for measurement of β-lactam residues in food and environment.@en