Prussian blue nanoparticles (PBNPs), also called nanozymes, are very attractive as an alternative to horseradish peroxidase in immunoassay development due to their simple and low-cost synthesis, stability and high catalytic activity. Today, there is a method for highly effective PBNP synthesis based on the reduction of an FeCl/K[Fe(CN)] mixture by hydrogen peroxide. However, there is a lack of research showcasing the use of these highly effective PBNPs for specific target detection in clinical settings, as well as a lack of comprehensive comparisons with conventional methods. To address this gap, we prepared diagnostic reagents based on highly effective PBNPs by modifying them using gelatin and attaching anti-C-reactive protein (CRP) monoclonal antibodies through cross-linking with glutaraldehyde. As a result, a solid-phase colorimetric immunoassay in a sandwich format (nanozyme-linked immunosorbent assay [NLISA]) using highly effective PBNPs as a label for CRP detection has been demonstrated for the first time. The assay demonstrated a detection limit of 21.8 pg/mL, along with acceptable selectivity, precision (CV < 25%) and accuracy (the recovery index was within acceptable limits (75-125%) for LLOQ /ULOQ range. The analytical performance of this method is on par with sensitive assays developed in the last 5 years. Notably, the results obtained from NLISA align with those from an immunofluorescence assay conducted by a certified clinical laboratory. Furthermore, this study underscores the technological challenges involved in constructing an analysis that necessitate further exploration.