The application of selenium nanoparticle (SeNP)-based fertilizers can cause SeNPs to enter the soil environment. Considering the possible transformation of SeNPs and the species-dependent toxicity of selenium (Se), accurate analysis of SeNPs and other Se species present in the soil would help rationally assess the potential hazards of SeNPs to soil organisms. Herein, a novel method for speciation of SeNPs and other Se species in soil was established. Under the optimized conditions, SeNPs, selenite, selenate, and seleno amino acid could be simultaneously extracted from the soil with mixtures of tetrasodium pyrophosphate (5 mM) and potassium dihydrogen phosphate (1.2 μM), while inert Se species (mainly metal selenide) remained in the soil. Then, extracted SeNPs can be effectively captured by a nylon membrane (0.45 μm) and quantified by inductively coupled plasma mass spectrometry (ICP-MS). Other extracted Se species can be separated and quantified by high-performance liquid chromatography coupled with ICP-MS. Based on the difference between the total Se contents and extracted Se contents, the amount of metal selenide can be calculated. The limits of detection of the method were 0.02 μg/g for SeNPs, 0.05 μg/g for selenite, selenate, and selenocystine, and 0.25 μg/g for selenomethionine, respectively. Spiking experiments also showed that our method was applicable to real soil sample analysis. The present method contributes to understanding the speciation of Se in the soil environment and further estimating the occurrence and application risks of SeNPs.