Immobilization of Lactobacillus rhamnosus ATCC7469 in poly(vinyl alcohol)/calcium alginate (PVA/Ca-alginate) matrix using "freezing-thawing" technique for application in lactic acid (LA) fermentation was studied in this paper. PVA/Ca-alginate beads were made from sterile and non-sterile PVA and sodium alginate solutions. According to mechanical properties, the PVA/Ca-alginate beads expressed a strong elastic character. Obtained PVA/Ca-alginate beads were further applied in batch and repeated batch LA fermentations. Regarding cell viability, L. rhamnosus cells survived well rather sharp immobilization procedure and significant cell proliferation was observed in further fermentation studies achieving high cell viability (up to 10.7 log CFU g-1) in sterile beads. In batch LA fermentation, the immobilized biocatalyst was superior to free cell fermentation system (by 37.1%), while the highest LA yield and volumetric productivity of 97.6% and 0.8 g L-1 h-1, respectively, were attained in repeated batch fermentation. During seven consecutive batch fermentations, the biocatalyst showed high mechanical and operational stability reaching an overall productivity of 0.78 g L-1 h-1. This study suggested that the "freezing-thawing" technique can be successfully used for immobilization of L. rhamnosus in PVA/Ca-alginate matrix without loss of either viability or LA fermentation capability.