Antibiotic resistance, encoded via particular genes, has become a major global health threat and substantial burden on healthcare. Hence, the facile, low-cost, and precise detection of antibiotic resistance genes (ARGs) is crucial in the realm of human health and safety, especially multiplex sensing assays. Here, a smart pH-regulated switchable photoelectrochemical (PEC) bioassay has been created for ultrasensitive detection of two typical subtypes of penicillin resistance genes bla -CTX-M-1 (target 1, labeled as T DNA1 ) and bla -TEM (target 2, labeled as T DNA2 ), whereby pH-responsive antimony tartrate (SbT) complex-grafted silica nanospheres are ingeniously adopted as signal DNA1 tags (labeled as S DNA1 -SbT@ SiO 2 NSs). The operations of the PEC bioassay depend on the switchable dissociation of the pH-responsive S DNA1 -SbT@SiO 2 NSs complex under the external pH stimuli, thus initiating the pH-regulated release of ions pre-embedded in sandwich-type DNA nanoassemblies. At acidic conditions, the dissociation of S DNA1 tags (ON state) triggers the release of the embedded SbO + . Under alkaline conditions, the dissociation of S DNA1 tags is inhibited (OFF state). The detection of T DNA2 was achieved via DNA hybridizationtriggered metal ion release. The unwinding of the introduced hairpin T-Hg 2+ -T fragment, hybridized with the second anchored signal DNA (S DNA2 ), ignites the release of Hg 2+ . The released SbO + or Hg 2+ ions would trigger the formation of Sb 2 S 3 /ZnS or HgS/ ZnS heterostructure through ion-exchange with the photosensitive ZnS layer, giving rise to the amplified photocurrents and eventually realizing the ultrasensitive detection of penicillin resistance genes subtypes, bla -CTX-M-1 and bla -TEM . The as-fabricated pHregulated PEC bioassay, smartly integrating the pH-responsive intelligent unit as S DNA tags, pH-regulated release of embedded ions, and the subsequent ion-exchange-based signal amplification strategy, exhibits high sensitivity, specificity, low-cost, and ease of use for multiplex detection of ARGs. It can be successfully used for measuring bla -CTX-M-1 and bla -TEM in real E. coli plasmids, demonstrating great promise for developing a new class of genetic point-of-care devices.