A novel automated integrated platform for quantitative proteome analysis was established with a combination of online digestion of proteins and in situ¹⁸O labeling by an immobilized enzyme reactor (IMER); digests were captured and desalted by a C18 trap column, and peptides were analyzed by nanoRPLC-ESI-MS/MS. Bovine serum albumin (BSA) was used to evaluate the performance of the developed platform. Compared with traditional offline methods, not only the digestion and labeling time was shortened from 36 h to just 1 h, but also the labeling efficiency was improved from 95% to 99%. Furthermore, the back-exchange from ¹⁸O to ¹⁶O could also be efficiently avoided by the use of IMER. The platform was further evaluated by the quantitative analysis of 100 ng ¹⁸O and ¹⁶O online labeled yeast sample with a mixing ratio of 1 : 1, and the results showed significantly improved sensitivity and reproducibility, as well as improved quantitative accuracy than offline method. With these advantages, the integrated platform was finally applied to the quantitative profiling of 100 ng proteins extracted from two mouse hepatocarcinoma ascites syngeneic cell lines with high and low lymph node metastases rates, and ten differentially expressed proteins were successfully found, most of which were related to tumorigenesis and tumor metastasis. All these results demonstrate that the developed integrated platform can provide a new way for high efficiency ¹⁸O labeling and the quantitative analysis of trace amounts of sample with high accuracy and high reproducibility.