Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the crosslinking DNA for selective target recognition. Hydrogel response to target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H₂O₂ to O₂. Then, the generated O₂ in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings.