<p>An&#xa0;in situ nanozyme assembly amplification strategy is reported based on the programmable bioaffinity-driven assembly of streptavidin-Pd@Pt and biotin-Pd@Pt nanozymes, enabling ultrasensitive colorimetric immunoassay of protein biomarkers at sub-femtomolar levels. In this design, streptavidin-Pd@Pt nanozymes are first anchored onto the immunocomplexes through standard biotinylated antibody recognition, followed by the recruitment of biotin-Pd@Pt nanozymes <i>via</i> strong streptavidin-biotin interactions. This stepwise and localized assembly process induces the formation of high-density Pd@Pt nanozyme clusters directly on the target recognition sites, dramatically increasing the local nanozyme concentration and catalytic accessibility. Benefiting from the exceptional peroxidase-like catalytic activity of the precisely engineered Pd@Pt nanozymes, together with the assembly-induced signal multiplication effect, the resulting nanozyme assemblies generate substantially amplified colorimetric signals. As a proof of concept, the proposed immunoassay enables quantitative detection of interleukin-10 (IL-10, selected as a model protein biomarker) with a detection limit as low as 10&#xa0;fg mL^− 1 (0.54 fM), achieving approximately two orders of magnitude improvement in sensitivity compared with the conventional HRP-based colorimetric immunoassay. This in situ nanozyme assembly amplification strategy provides a simple yet powerful route to transcend the sensitivity limits of traditional colorimetric immunoassays, offering a versatile and readily translatable platform for ultrasensitive biomarker detection.</p> Graphical abstract <p></p>