This study compares the performance of novel fluorescent nanobiosensors and liquid chromatography–mass spectrometry (LC-MS) for detecting organophosphorus pesticide residues in Angelica sinensis. Key parameters such as recovery rate and relative standard deviation (RSD) were evaluated to identify a preferable detection method. The results show that the fluorescent nanobiosensor offers superior sensitivity, whereas LC-MS excels in accuracy and quantitative analysis. For phoxim, the GQDs@GSH sensor achieved an LOD of 0.075 μmol·L⁻¹, with recoveries of 97.65–100.62% (RSD < 3.35%). For glyphosate, the PDOA/Cu²⁺ sensor achieved an LOD of 1.8 nmol·L⁻¹, and the AgNCs sensor achieved an LOD of 21 nmol·L⁻¹, with recoveries of 91.30–105.34% (RSD < 3.35%). The originality of this work is threefold and does not claim de novo synthesis of unknown materials: (i) it is the first validation of three existing fluorescent nanosensors (GQDs@GSH, PDOA/Cu²⁺, and AgNCs) specifically for organophosphorus pesticide detection in the complex medicinal plant matrix Angelica sinensis; (ii) it provides the first systematic comparison of these nanosensors against the pharmacopeia-standard LC-MS method in this matrix, delineating their complementary advantages; (iii) it is the first assessment of their phytotoxicity in Angelica sinensis seedlings, demonstrating biocompatibility and potential for in planta imaging.