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Abstract: Accurate profiling of metabolites in biofluids provides a feasible approach to disease diagnosis and therapeutic evaluation in clinics. However, current material‐based analytical platforms for in vitro metabolite detection often requires tedious sample pretreatment. To address this issue, herein, we report an aptamer‐functionalized fractal gold nanoflower (Apt‐AuNF)‐based sensing platform for metabolite analysis. A series of fractal AuNFs with tunable nanoshell structures was synthesized using DNA‐engineered strategy, and optimized Apt‐AuNFs were demonstrated to enable efficient trap of target analytes with ∼5‐fold enhanced enrichment, thus achieving selective analysis of metabolites in complex biofluids without requirement of pre‐enrichment and purification. We applied Apt‐AuNF‐assisted laser desorption/ionization mass spectrometry for disease diagnosis, identifying diabetic patients through daily monitoring and glucose quantification in serum. This work provides a guideline to design materials for high‐performance metabolic analysis and precise disease diagnosis. [ABSTRACT FROM AUTHOR]