Microfluidic techniques are widely applied in biomolecular analysis and disease diagnostic assays. While the volume of the sample that is directly used in such assays is often only femto-to microliters, the "dead volume" of solutions supplied in syringes and tubing can be much larger, even up to milliliters, increasing overall reagent use and making analysis significantly more expensive. To reduce the difficulty and cost, we designed a new chip using a low volume solution for analysis and applied it to obtain real-time data for protein-protein interaction measurements. The chip takes advantage of air/aqueous two-phase droplet flow, on-chip rapid mixing within milliseconds, and a droplet capture method, that ultimately requires only 2 μL of reagent solution. The interaction is analyzed by particle diffusometry, a nonintrusive and precise optical detection method to analyze the properties of microparticle diffusion in solution. Herein, we demonstrate on-chip characterization of human immunodeficiency virus p24 antibody-antigen protein binding kinetics imaged via fluorescence microscopy and analyzed by PD. The measured kon and koff are 1 × 106 M-1 s-1 and 3.3 × 10-4 s-1, respectively, and agree with independent measurement via biolayer interferometry and previously calculated p24-antibody binding kinetics. This new microfluidic chip and the protein-protein interaction analysis method can also be applied in other fields that require low-volume solutions to perform accurate measurement, analysis, and detection.