Different commercially available culture media have been in use for the isolation and identi cation of microorganisms for many years. However, the high cost and limited availability culture media has been a problem in the local market of Tigray, Ethiopia that hinder researchers from carrying out their study. Therefore, the present study aimed to develop locally available cheap plant-based culture medium from cactus pear (Opuntia cus-indica) cladodes extract for the growth of Lactobacillus species. For this purpose, free external injuries of cactus pear cladode samples were collected from the vicinity of Adigrat, Tigray, Ethiopia. For the test two Lactobacillus species (Lactobacillus reuteri and Lactobacillus mali) were used. The growth of Lactobacillus species on cactus pear cladode agar and broth (4:100, 4:200 and 4:300 w/v) were evaluated and compared with de Man, Rogosa, and Sharpe (MRS) medium. All prepared cactus pear cladode agar (CPCA) media were transparent and free from particles and turbidity. Both tested Lactobacillus species were produced small, white and round colonies on CPCA at 72 hours without bad odor and any pigments. Exponential growth was observed in cactus pear cladode broth (4:200 w/v) in short time than other cactus pear cladode broth (4:100 and 4:300 w/v) medium. When compared with MRS medium (control), the colony counts of Lactobacillus species in cactus pear cladode broth (4:200 w/v) medium did not differ signi cantly (P < 0.05). The present study clearly showed that the cactus pear cladode (4:200 w/v) medium could be used as alternative culture medium to support growth of Lactobacillus species.