The second-order scattering (SOS) phenomenon of the interaction of Pd nanoparticles with protein was reported and a simple, sensitive, palladium nanoparticle-based assay for trace amount of protein with SOS technique was developed. The SOS intensities were significantly enhanced due to the interaction of Pd nanoparticles with bovine serum albumin (BSA) or human serum albumin (HSA) at pH 3.5 or 4.0, respectively. The maximum SOS peak appeared at 260/520 nm (λex/λem). The optimal experiment conditions, affecting factors and the influence of some coexisting substances were checked. The SOS intensity increased proportionally with the increase of Pd concentration below 3.0 × 10?5 mol·L?1, while declined gradually above 4.0 × 10?5 mol·L?1. BSA within the range of 0.01-2.6 μg·mL?1 and HSA of 0.01-1.7 μg·mL?1 can be detected with this method and the detection limits were 2.3 and 11.2 ng·mL?1, respectively. The method was successfully applied to the quantitative detection of total protein content in human serum samples with the maximum relative standard deviation (RSD) lower than 2.6% and the recoveries over the range of 99.5-100.5%.