Hyperglycemia and diabetes are widely recognized as prevalent metabolic disorders, often associated with various complications, including hepatic and renal dysfunction as well as cardiovascular damage. Lactic acid bacteria (LAB) strains have been proposed as potential agents for ameliorating hyperglycemia without causing adverse effects. In this study, we established a multi-target, comprehensive in vitro methodology to screen LAB strains with enhanced potential against hyperglycemia, using Lactobacillus rhamnosus(LGG) as a reference strain. We initially selected 43 strains isolated from the human gastrointestinal tract based on their alpha-glucosidase inhibitory activity; subsequently, their capacity to inhibit alpha-amylase was evaluated through alpha-amylase inhibition assays. Furthermore, insulin sensitization was assessed via tyrosine phosphatase 1B inhibition, along with antioxidant capabilities. To thoroughly evaluate the efficacy of LAB strains in improving abnormal glucose metabolism, we developed an insulin-resistant HepG2 cell model. Principal component analysis revealed that NCUH012072 (Lacticaseibacillus paracasei), NCUH003081 (Limosilactobacillus fermentum), NCUH064006 (Limosilactobacillus reuteri), and two strains of NCUH001099 (Lactiplantibacillus plantarum) exhibited superior hypoglycemic properties. Concurrently, these LAB strains demonstrated robust acid resistance and bile salt tolerance while showing neither hemolytic activity nor antibiotic resistance. These findings suggested that NCUH012072 (Lacticaseibacillus paracasei), NCUH003081 (Limosilactobacillus fermentum), and NCUH064006 (Limosilactobacillus reuteri) are promising probiotic candidates for glycemic control.