We have developed a sensitive, specific, rapid and low cost pico-liter microsphere based platform for detection and quantification of human anti-tetanus immunoglobulin G. In this method, a biological sample, biosensing microspheres, and fluorescently labeled detection antibodies are co-encapsulated to capture the analyte on the surface of the microsphere in microfluidic pL-sized droplets. Using our platform and commercially available antibodies, we quantified the anti-tetanus antibodies content in human serum.