The hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver. It is a common malignant tumor in clinic. The main reason for its high mortality is its early latency. Therefore, how to accurately determine and test the hepatocellular carcinoma in the early stage has a very positive significance for the treatment. It is an important method for the early diagnosis of the hepatocellular carcinoma to use aptamers specifically binding to hepatocellular carcinoma cells, which has good application prospects. In order to improve the efficiency of aptamer selection of tumor cells, our group designed and developed an automated instrument for the aptamer selection. In this paper, the method to separate bound aptamers from the surface of HepG2 cells in automated selection process was studied, and the feasibility of separating binding aptamers from the HepG2 cell surface using ultrapure water and the effect of different temperature environments on its isolation were discussed. Results of the real-time fluorescent PCR and flow cytometry showed that ultrapure water could be used to isolate bound HepG2 cells and aptamers, and the concentration of the aptamers increased with the rise of the temperature between 25 and 80 degrees Celsius. This result will contribute to the improvement on the efficiency of automated selections for aptamers corresponding to HepG2 cells.