Expression of TGF-β1 and miR-99a in patients with early spontaneous abortion and correlation with hormone levels during pregnancy were investigated. A total of 70 pregnant women with early spontaneous abortion diagnosed in Jining No. 1 People's Hospital from February 1, 2015 to May 1, 2018 were selected as the study group, and 83 normal pregnant women who chose abortion for non-medical reasons in the same period as the control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect TGF-β1 and the levels of serum β-HCG, progesterone and estrogen during pregnancy in the two groups, and RT-qPCR to detect the expression of miR-99a, and partial correlation analysis to analyze the correlation of TGF-β1 and miR-99a with the levels of serum β-HCG, progesterone and estrogen in the study group of patients. Expression of β-HCG was significantly lower in the study group than that in the control group, with a statistically significant difference (P<0.001), and that of progesterone was significantly lower in the study group than that in the control group, with a statistically significant difference (P<0.001). Expression of estrogen was significantly lower in the study group than that in the control group, with a statistically significant difference (P<0.001). The partial correlation analysis indicated that the levels of serum β-HCG, progesterone and estrogen were positively correlated with TGF-β1 (r=0.944, 0.868, 0.869, P<0.001), negatively correlated with the expression level of miR-99a (r=−0.944, −0.892, −0.891, all P<0.001). miR-99a was highly expressed in the serum of patients with early spontaneous abortion, but TGF-β1 expression was low. The expression levels of the two factors are related to hormone levels during pregnancy, which are expected to be new candidate molecular diagnostic markers in the diagnosis of early spontaneous abortion.