Evolvulus alsinoides L. is a medicinal plant of the family Convolvulaceae. It is used traditionally for its medicinal properties. The present investigation was carried out to standardize an efficient in vitro protocol for callus induction and plant regeneration using leaf, nodal and internodal explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators like 2,4-D, NAA, BAP and IBA. Results showed that nodal explants gave the maximum 100 % callus induction on MS medium supplemented with 1.5 mg/L 2,4-D and 1.5 mg/L NAA, and showed the highest callogenic response. Callus initiation was observed after about 10 days. Substantial callus development occurred during subsequent culture. The results show the potential of in vitro tissue culture for propagation and conservation of E. alsinoides and as a basis for further studies on the production of its valuable secondary metabolites.