Conventional diagnostic methods for biomarker detection often require invasive procedures and exhibit limited reproducibility and sensitivity. In this study, the turbulence-enhanced microneedle immunoassay platform (TMIP) was designed to enhance the performance and accuracy of biomarker detection in skin interstitial fluid (ISF). TMIP combines a bullet-shaped microneedle (MN) array for minimally invasive biomarker capture, a microfluidic device for MN-mediated immunoassay process simplification, and a star-shaped magnetic stirrer tool (MST) to facilitate efficient washing. By targeting S100 calcium-binding protein B (S100B), a diagnostic biomarker for melanoma, TMIP demonstrated substantial improvements in reproducibility, reducing signal deviations by up to 55 % compared to manual operation. The application of nanoporous MNs (NPMNs) achieved a low detection limit of 20 pg/mL with a high linearity (R = 0.9758). Validation using a gelatin phantom mimicking human skin confirmed TMIP's ability to achieve improved reproducibility and sensitivity. Furthermore, TMIP successfully detected S100B with high reproducibility in both the phantom (R = 0.97523) and melanoma-expressing mice within a rapid incubation time of 1 min. TMIP enables the detection of biomarkers with remarkable reproducibility and sub-nanogram sensitivity by simplifying the analysis process and enhancing reagent washing through turbulence. These features suggest that TMIP has the potential to serve as an efficient and reliable tool for biomarker detection in skin ISF.