The development of a novel, highly sensitive electrochemical sensor for ASP based on an alizarin red S-functionalized zeolitic imidazolate framework-8 (ALZ@ZIF-8) nanocomposite. The nanocomposite was synthesized via a one-pot approach and fabricated on a carbon paste electrode (ALZ@ZIF-8/CPE). Its interaction with ASP was elucidated through density functional theory (DFT) calculations and electrochemical analysis, while its structure was confirmed using SEM, XRD, FT-IR, UV-Vis, and spectrofluorimetric techniques. Under optimized conditions, a square-wave voltammetric (SWV) method was developed and rigorously validated. The sensor demonstrated a wide linear range from 0.01 to 14.0 µM, with exceptional sub-micromolar sensitivity (detection limit: 3.45 nM; quantification limit: 10.46 nM). The method showed high accuracy (recoveries: 99.26–101.62%) and precision (relative standard deviation, RSD ≤ 1.01%). Successful application to spiked human plasma yielded recoveries ≥ 98.84% with RSDs ≤ 1.61%, confirming practical utility. This work presents the first application of the ALZ@ZIF-8 nanocomposite in analysis. The proposed sensor offers a promising and adaptable platform for the sensitive detection of ASP and other target molecules in complex biological media.