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In accurately diagnosing Alzheimer's disease (AD) and distinguishing AD from other dementia, the concentration ratio of amyloid-beta 42 (Aβ 42 ) to Aβ 40 is more reliable than the concentration of Aβ 42 alone. For the multiplex PEC assay, generating an independent photocurrent of multiple targets on a single interface is a great challenge. Herein, an i-motif-based switchable sensing approach is proposed to construct a pH-regulated multiplex PEC immunosensor for Aβ 42 and Aβ 40 by using Bi-TBAPy as an efficient photoactive cathode material. An independent photocurrent signal of Aβ 42 and Aβ 40 is produced through the regulation of the electron-transfer tunneling distance by a pH-dependent configuration transition of the i-motif DNA. In a 96-well plate, immunological recognition of Aβ 42 (or Aβ 40 ) coupled with an enzymatic catalytic reaction produces an acidic (or alkaline) lysis solution, which triggers the formation and unravelment of the i-motif structure. The above configuration transition regulates the distance between Au NPs labeled SH-DNA and Bi-TBAPy, leading to PEC signal switching. Smart integration of the pH-responsive switchable DNA probe with a high-efficiency photocathode enables the precise monitoring of Aβ 42 and Aβ 40 at a single interface in a wide detection range (10 fg/mL ∼ 1 μg/mL and 1 pg/mL ∼ 1 μg/mL) with detection limit of 4.5 fg/mL and 0.52 pg/mL, respectively. The proposed i-motif-based switchable sensing strategy paves a new avenue for a multiplex PEC assay on a single interface, showing great prospects in bioanalysis and early disease diagnosis.