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Lactic acid bacteria strains (LAB) are regarded as safe food-grade microorganism and have been used in fermented food production and preservation for a long history. Some of the LAB strains are considered as probiotics and have the potential to regulate diabetes. In order to screen potential hypoglycemic probiotic strains in vitro, 10 Lactobacillus strains isolated from traditional dairy products were determined for the inhibitory activity against α-glucosidase from porcine intestine. The basic features of probiotics, including gastrointestinal fluid tolerance and cell adhesion, were also investigated. Some strains were selected for further study of their effects on α-glucosidase activity and gene expression level by Transwell model established by Caco-2 cells. The results showed that all the 10 strains showed different degrees of α-glucosidase inhibitory activity, which peaked at the logarithmic or stationary phase of the strains growth, and then decreased. The evaluation of the basic features of probiotics suggested that three strains with higher α-glucosidase inhibitory activity showed tolerance to the simulated gastrointestinal fluid and strong HT-29 cell adhesion. The Caco-2 cells model showed that Lactobacillus strains could both inhibit α-glucosidase activity and its mRNA expression. The selected three Lactobacillus strains, including Lactobacillus paracasei strains L14 and Z3-11, and Lactobacillus plantarum strain NL42 displayed high α-glucosidase activity and good probiotic properties and may serve as potential probiotics for the auxiliary management of diabetes.