Developing the portable CRP detection technologies that are suitable for point-of-care (POC) and primary care management is of utmost importance, and advancing the electrochemical immunosensors hold promise for POC implementation. Nevertheless, non-specific adsorption of numerous interfering proteins in complex biological media contaminates immunosensors, thereby restricting the reliability in detection efficacy. In this study, a three-dimensional flower-leaf shape amyloid bovine serum albumin/gold nanoparticles/polyaniline (AL-BSA/AuNPs/PANI) coating on the surface of the electrode was developed, which demonstrated strong anti-adsorption properties against bovine serum albumin, plasma, and cells. The immunosensor exhibited a good linear relationship to CRP response, featuring a detection limit of 0.09 μg/mL, consistent with clinical reference range. In addition, the CRP immunosensor demonstrated excellent specificity in other inflammation-related proteins and commendable anti-interference performance for CRP detection in plasma and whole blood tests. Importantly, by combining the development of a USB flash disk-type portable electrochemical workstation with a reagent-free mode, the developed CRP electrochemical immunosensor delivered ideal results in clinical samples. The anti-fouling performance, sensitivity and specificity of the immunosensor, as well as its flexible test modes in clinical samples, provide important scientific basis for developing POC detection technologies of vital biomarkers in complex biological media.