This study aims to prepare gastric cancer stem-like cells(GCSCs) using a serum-free suspension culture, then identify it preliminarily, and observe the expression level of Trop2. Serum-free DMEM/F12 medium and low-adhesion dish were used for suspended culture of gastric cancer (GC) cell lines MGC803. The morphological characteristics of cell spheres were observed by optical microscope; the positive rates of the CD44, CD54, EpCAM and Trop2 in MGC803 and MGC803-spheres were detected by FACS; the changes of the cell cycle in the MGC803-spheres were explored by FACS and compared with that in MGC803; the mRNA level of cancer stem cells(CSCs) regulatory genes and Trop2 in MGC803-spheres and MGC803 were detected by qRT-PCR. MGC803-spheres formed after MGC803 was cultured in serum-free medium for about 14 days; the levels of CD44, CD54, EpCAM and Trop2 in MGC803-spheres group were higher than those in MGC803 group (P<0.05). The G1 phase of cell cycle in the MGC803-spheres group was obviously lower than that in MGC803 group, and the S phase of the cell cycle in MGC803-spheres group was obviously higher than that in the MGC803 group (P<0.05). The mRNA level in the CSCs regulatory genes (Oct4, Snail, Nanog) and Trop2 in MGC803-spheres group were much higher than those in MGC803 group (P<0.05). MGC803-spheres could form in serum-free and suspension culture condition. The type of cells has the characteristic of CSCs, which appears in a higher proliferation state and over expression CSCs regulating genes and Trop2.