Fluorescent pH biosensors have gained importance owing to their low cost utilization in real time monitoring of biological and food samples in comparison to conventional pH meters. The research reports a novel method of ultrasonic atomization for developing a fluorescent pH sensor for real-time analysis made of Fluorescein isothiocyanate (FITC)-dextran/FITC-dextran-Tris (2, 2'-bipyridyl) dichlororuthenium (II) hexahydrate as indicator and reference fluorophores, respectively. The process of ultrasonic atomization ensures formation of monodisperse dye immobilized alginate microspheres ensuring efficient pH sensing. The developed biosensor was tested on milk samples, which has a short life span and shows a significant fall in pH with time due to microbial spoilage. The proposed biosensor showed a linear range of pH 4-8 (R2 between 0.96-0.99 for different single/dual fluorophore biosensors) which suitably cover the pH of milk during the entire storage period and spoilage. The % recovery for predicted pH falls between 90-110% compared against standard pH meter, indicating a good accuracy of estimation and low turnaround time (10 min). Thus, real-time monitoring using fluorescent pH biosensor for milk samples may profoundly improve the economics of losses occurring in processing and storage with capability of in-package continuous quality assessment.