Instant and accurate detection of 3‐nitrotyrosine (3‐NT) biomarker is an important but challenging task in clinical diagnosis, treatment, and monitoring of diseases. Herein, this work reported a Zn (II)–based metal–organic framework (MOF) {[Zn2(BDPT)(DIMB)]}n, denoted as Zn‐BDPT, synthesized by solvothermal method using bis(3,5‐dicarboxyphenyl)terephthalamide (H4BDPT) and 1,4‐di(1H‐imidazol‐1‐yl)butane (DIMB). Neighboring [Zn (COO)2] clusters were connected by BDPT4− units and DIMB to form a (2,4,4)‐connected three‐dimensional (3D) architecture. X‐ray powder diffraction (PXRD) and thermogravimetric (TGA) experiments indicated that Zn‐BDPT had good purity and stability. Zn‐BDPT could selectively and rapidly recognize 3‐NT in an aqueous environment. For rapid economic detection of 3‐NT in actual samples, mixed‐matrix membranes (MMMs) based on Zn‐BDPT were successfully prepared and named Zn‐BDPT@PMMA. The addition of 3‐NT under the UV lamp displayed an evident visible fluorescence quenching effect. Quantitative titration analysis of Zn‐BDPT@PMMA in real serum sample obtained the Ksv value of 1.13 × 104 M−1 and a limit of detection (LOD) of 0.17 μM. This report demonstrated that MOF‐based MMMs could be used as ideal candidate for luminescent probe in actual application.