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In this work, an ant nest structured porous carbon nanosphere had been developed for the recognition detection of the atropine (ATP) enantiomers Dhyoscyamine (D-HSM) and L-hyoscyamine (L-HSM). Firstly, Fe-based organic framework was used as the substrate, and Cu ions and sulfur ions were separately introduced to obtain CuFeS 2 with ant nest structure by hydrothermal incubation. Then CuFeS 2 /C porous nanospheres (PNSs) were obtained by high-temperature calcination. The composite-modified electrode exhibited superior electrochemical performance for L-HSM due to the synergistic effect of CuFeS 2 cubic crystals and porous carbon, which has the high specific surface area of the ant nest structure. In addition, the molecularly imprinted polymer (MIP) about L-HSM formed with sulfonated-β-cyclodextrin (S-β-CD) and L-arginine (L-Arg) by cyclic voltammetry showed strong chiral recognition of D/L-HSM (ATP). Therefore, a novel electrochemical sensor was constructed based on CuFeS 2 /C PNSs and MIP to detect L-HSM by differential pulse voltammetry. Under the optimal conditions, the peak current density of L-HSM showed a good linearity in the concentration range of 0.02-4.6 μM with LOD and LOQ of 0.45 and 1.5 nM, respectively. The oxidation peaks of L-HSM and D-HSM were successfully identified from the racemic ATP, and the oxidation peak potential difference (ΔE p ) between them was 0.138 V. In conclusion, the sensor showed excellent reproducibility, repeatability, and stability and had been applied to the determination of L-HSM in human serum, saliva, and ATP sulfate tablets with satisfactory results.