<p>A magnetic nanoparticle-based chemiluminescent immunoassay (MNPs-CLIA) for serum surfactant protein D (SP-D) quantification and evaluation of its clinical diagnostic value has been developed. Recombinant SP-D protein was expressed and purified, monoclonal antibodies (mAbs) were generated and screened, and the MNPs-CLIA system was optimized and validated. Analytical performance was systematically evaluated. The clinical diagnostic value was assessed by measuring serum SP-D levels in 338 specimens. The recombinant SP-D protein (molecular weight 43 kDa) matched theoretical predictions. Hybridoma cells stably secreting SP-D-specific mAbs were obtained, and paired antibodies were selected based on epitope recognition and binding affinity. The MNPs-CLIA achieved a detection limit of 0.121 ng/mL and a linear range of 1–1000 ng/mL, meeting precision, accuracy, and specificity standards for in vitro diagnostics and showing high consistency with ELISA results (R² = 0.9976). Serum SP-D levels were significantly elevated in chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), and silicosis patients (P &lt; 0.05), with ROC-AUC values of 0.959, 0.946, and 0.956, respectively, demonstrating excellent diagnostic performance. The developed MNPs-CLIA enables rapid, sensitive, and specific quantitative detection of serum SP-D, demonstrating suitability for clinical application with high diagnostic value for multiple pulmonary diseases.</p> Graphical Abstract <p></p>