Developing an enhanced diagnosis using biosensors is important for the treatment of patients before disease complications arise. Improving biosensors would enable the detection of various low-abundance disease biomarkers. Efficient immobilization of probes/receptors on the sensing surface is one of the efficient ways to enhance detection. Herein, we introduced the pre-alkaline sensing surface with amine functionalization for capturing gold nanoparticle (GNP) conjugated to human blood clotting factor IX (FIX), and we demonstrated the excellent performance of the strategy. We have chosen the enzyme-linked immunosorbent assay (ELISA) and the interdigitated electrode (IDE), which are widely used, to demonstrate our method. The optimal amount for silanization has been found to be 2.5%, and 15-nm-sized GNPs are ideal and characterized. The limit of FIX detection was attained with ELISA at 100 pM with the premixed GNPs and FIX, which shows 60-fold improvement in sensitivity without biofouling, as compared to the conventional ELISA. Further, FIX was detected with higher specificity in human serum at a 1:1280 dilution, which is equivalent to 120 pM FIX. These results were complemented by the analysis on IDE, where improved detection at 25 pM was achieved, and FIX was detected in human serum at the dilution of 1:640. These optimized surfaces are useful for improving the detection of different diseases on varied sensing surfaces.