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Agaricus bisporus mushroom biomass contains a lectin, ABL, with remarkable specificity for lactose biorecognition; in this work, this feature was explored to develop a photoelectrochemical biosensor. The high lectin activity found in saline extracts of this macrofungus (640 HU mL−1), even at critical pH values (4–10) and temperatures (20–100 °C), allowed its direct use as an ABL source. Theoretical and experimental evidence revealed favorable electrostatic and biocompatible conditions to immobilize ABL on a poly(methylene blue)/fluorine-doped tin oxide-coated glass platform, giving rise to the ABL/PMB/FTO biosensor. The conducting polymer added further photoactivity to the device, allowing the identification of lectin–carbohydrate interactions with even greater sensitivity. The dose–response curves studied by electrochemical impedance spectroscopy showed a sigmoidal profile that was well-fitted by Hill’s equation, expanding the working dynamic range (15–540 nmol L−1 lactose; 20.2 pmol L−1 detection limit) and avoiding undesirable sample dilution or preconcentration procedures. Under the optimized photoelectrochemical conditions, the ABL/PMB/FTO biosensor showed remarkable signal stability, accuracy, specificity, and selectivity to analyze lactose in commercial food products. This research raises interest in ABL-based biosensors and the added value of the crude Agaricus bisporus extract toward the development of greener and more sustainable biotechnological approaches.