This study presents the development of stannous chloride (SnCl₂)-modified glass substrates for biomolecule immobilization and their application in fabricating sensor chips for label-free interferometric biosensors. The glass modification process was optimized, identifying a 5% SnCl₂ concentration, a 45 min reaction time, and a 150 °C drying temperature as conditions for efficient protein immobilization. Based on the SnCl₂-modified glass substrates and label-free spectral-phase interferometry, a biosensor was developed for the detection of aflatoxin B1 (AFB1)—a highly toxic and carcinogenic contaminant in agricultural products. The biosensor realizes a competitive immunoassay of a remarkable detection limit as low as 26 pg/mL of AFB1, and a five-order dynamic range. The biosensor performance was validated using real corn flour samples contaminated with Aspergillus flavus. The proposed approach not only provides a powerful tool for AFB1 detection for food safety monitoring but also demonstrates the potential of SnCl₂-modified substrates as a versatile platform for the development of next-generation biosensors.