Pressure-based bioassays incorporating biomolecular recognition with a catalyzed gas-generation reaction have been developed for gas biosensors, but most involve poor sensitivity and are unsuitable for routine use. Herein we design an innovative gas pressure-based biosensing platform for the detection of Kanamycin (Kana) on polyaniline nanowires-functionalized reduced graphene oxide (PANI/rGO) framework by using platinum nanozyme-catalyzed gas generation. The signal was amplified by coupling with catalytic hairpin assembly (CHA) and strand-displacement amplification (SDA). Upon target Kana introduction, the analyte initially triggered a SDA reaction between hairpin DNA1 and hairpin DNA2, and then induced CHA conjugation between magnetic bead-labeled hairpin DNA3 (MB-H3) and platinum nanoparticle-labeled hairpin DNA4 (Pt-H4) to form a three-dimensional network. Numerous platinum nanoparticles (peroxidase-like nanozymes) were carried over with magnetic beads to reduce hydrogen peroxide into oxygen. The as-produced gas compressed PANI/rGO frameworks (modified to polyurethane sponge, used as the piezoelectric materials) in a homemade pressure-tight device, thus causing the increasing current of PANI/rGO sponge thanks to its deformation. The change in the current caused by the as-generated gas pressure was determined on an electrochemical workstation. Under optimum conditions, PANI/rGO sponge exhibited outstanding compressibility, stable signal-waveform output, fast response and recovery time (≈109 ms), and the current increased with the increasing Kana concentration within a dynamic working range of 0.2-50 pM at a detection limit of 0.063 pM. Good reproducibility, specificity, and acceptable precision were acquired for Kana analysis. In addition, the accuracy of this method was monitored to evaluate real milk samples with the well-matched results obtained by using the referenced Kana ELISA kit.