Low molecular weight proteins (LMWPs) in the bloodstream participate in various biological processes and are closely associated with disease status, whereas identification of serous LMWPs remains a great technical challenge due to the wide dynamic range of protein components. In this study, we constructed an integrated LMWPs library by combining the LMWPs obtained by three enrichment methods (50% ACN, 20% ACN+20 mM ABC, and 30 kDa) and their fractions identified by data dependent acquisition method. With this newly constructed library, we comprehensively profiled LMWPs in serum using data-independent acquisition and reliably achieved quantitative results for 75% serous LMWPs. When applying this strategy to quantify LMWPs in human serum samples, we could identify 405 proteins on average per sample, of which 136 proteins were less than 30 kDa and 293 proteins were less than 65 kDa. Of note, pre- and post-operative gastric carcinoma (GC) patients showed differentially expressed serous LWMPs, which was also different from the pattern of LWMPs expression in healthy controls. In conclusion, our results showed that LMWPs could efficiently distinguish GC patients from healthy controls as well as between pre- and post- operative statuses, and more importantly, our newly developed LMWPs profiling platform could be used to discover candidate LMWPs biomarkers for disease diagnosis and status monitoring.