Objective:By using carboxylated agar magnetic beads as selection medium,we sought to use RT-PCR and target replacement subtractive SELEX technology from the serum of gastric cancer screening to obtain aptamers with high specificity and affinity. Methods:The target molecules were gastric cancer serum performed with centrifugal ultrafiltration(ultrafiltration 50 000),and the carriers were the carboxyled agar magnetic beads. Firstly,oligonucleotide library and anti-screening agar magnetic beads(normal serum binding)were bound,then supernatant was combined with screening magnetic beads (gastric binding serum). We washed away unbound oligonucleotide molecules,and oligonucleotide molecules binding on the magnetic beads were isolated and amplified. λ enzyme digestion method was performed to prepare ssDNA second library for 10 rounds of screening. The library of the 10th round was amplified to obtain dsDNA,then we used gel extraction kit recovering purified fragment of dsDNA and connected it with pMDTM18-T vector and transformed into E. coli DH5α competent cells. we selected positive cloning group and sequenced them to obtain aptamers sequence. Simultaneous determination of Kd values of gastric cancer serum aptamer was performed by flow cytometry. Results:After 10 rounds of selection,20 gastric cancer serum specific aptamers were screened. Conclusion:The specific detection showed that the Kd values of gastric cancer serum binding aptamers were at the nanomolar level,and No.5,7,16,17,18 aptamers can bind to gastric cancer serum with high specificity and affinity,and didn’t bind to normal human serum. It provides an experimental basis for the early diagnosis of gastric cancer.