Sample pretreatment is an indispensable part of the analysis of complex biological matrices to ensure the accuracy and reliability of analytical results, but it is rather difficult to develop a miniaturized and portable strategy to minimize matrix interference. In light of the difficulties resulted by interference from biological matrices and the short half-life of some constituents, this work introduces a combination of an adjuvant tracer with a high-efficiency and portable sample pretreatment device, the portable steam penetration gas membrane separation (PSPGMS) device, achieving analysis of complex biological samples under the interference of matrices. In this work, methanol was utilized as adjuvant to capture free ONOO- in living cells under UV irradiation for its low background signal and eco-friendliness; the PSPGMS device conducted all-in-one separation, absorption, and derivatization for the detection of formaldehyde generated by the oxidization between methanol and the products of ONOO- photolysis, achieving a linear response (R2 = 0.9919) across 2.0-20.0 μmol/L and a recovery rate of 87.4-115.0% in the analysis of rat hepatocytes. With a detection limit of 0.5 μmol/L, excellent portability, and integration, this strategy is well-suited for on-site rapid and visualized detection, independent of bulky and expensive instruments.