Monitoring of uric acid (UA) levels in biological samples is of great significance for human health, while the development of a simple and effective method for the precise determination of UA content is still challenging. In the present study, a two-dimensional (2D) imine-linked crystalline pyridine-based covalent organic framework (TpBpy COF) was synthesized using 2,4,6-triformylphloroglucinol (Tp) and [2,2'-bipyridine]-5,5'-diamine (Bpy) as precursors via Schiff-base condensation reactions and was characterized with scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), Powder X-ray diffraction (PXRD), Fourier transform infrared (FT-IR) spectroscopy, and Brunauer-Emmett-Teller (BET) assays. The as-synthesized TpBpy COF exhibited excellent visible light-induced oxidase-like activity, ascribed to the generation of superoxide radicals (O) by photo-generated electron transfer. TpBpy COF could efficiently oxidase the colorless substrate 3,3',5,5'-tetramethylbenzydine (TMB) into blue oxidized TMB (oxTMB) under visible light irradiation. Based on the color fade of the TpBpy COF + TMB system by UA, a colorimetric procedure was developed for UA determination with a detection limit of 1.7 μmol L. Moreover, a smartphone-based sensing platform was also constructed for instrument-free and on-site detection of UA with a sensitive detection limit of 3.1 μmol L. The developed sensing system was adopted for UA determination in human urine and serum samples with satisfactory recoveries (96.6-107.8%), suggesting the potential practical application of the TpBpy COF-based sensor for UA detection in biological samples.