Novel microparticles have generated growing interest in diagnostics for potential sensitivity and specificity in biomolecule detection and for the possibility to be integrated in a micro-system array as a lab-on-chip. Indeed, bead-based technologies integrated in microfluidics could speed up incubation steps, reduce reagent consumption and improve accessibility of diagnostic devices to non-expert users. To limit non-specific interactions with interfering molecules and to exploit the whole particle volume for bioconjugation, hydrogel microparticles, particularly polyethylene glycol-based, have emerged as promising materials to develop high-performing biosensors since their network can be functionalized to concentrate the target and improve detection. However, the limitations in positioning, trapping and mainly fine manipulation of a precise number of particles in microfluidics have largely impaired point-of-care applications. Herein, we developed an on-chip sandwich immunoassay for the detection of human immunoglobulin G in biological fluids. The detection system is based on finely engineered cleavable PEG-based microparticles, functionalized with specific monoclonal antibodies. By changing the particle number, we demonstrated tuneable specificity and sensitivity (down to 3 pM) in serum and urine. Therefore, a controlled number of hydrogel particles have been integrated in a microfluidic device for on-chip detection (HyPoC) allowing for their precise positioning and fluid exchange for incubation, washing and target detection. HyPoC dramatically decreases incubation time from 180 minutes to one minute and reduces washing volumes from 3.5 ml to 90 μL, achieving a limit of detection of 0.07 nM (with a dynamic range of 0.07-1 nM). Thus, the developed approach represents a versatile, fast and easy point-of-care testing platform for immunoassays.