Objective: An efficient in vitro protocol was established during the development of polyembryony in the spore derived gametophyte of Pteris tripartita Sw. Methods: Sterilized spores were germinated in half strength Murashige and Skoog (MS) basal media to produce gametophyte at in vitro condition. Three month old gametophytes were sub-cultured in half strength MS medium with 6-benzylaminopurine (BAP). All the inoculated cultures were incubated and noticed every month for polyembryony. Results: Multiple numbers (Di, Tri, Tetra, Hexa, and Octa) of the juvenile sporophyte per spore derived gametophyte were observed at 3 mg/L of BAP in half strength MS basal media, which augmented with 30% sucrose. Hexa polyembryony or juvenile sporophytes was noticed on both 3 and 4 mg/L of BAP, which developed 1.00 and 0.76 cm lengths, respectively. At 4 mg/L of BAP in MS culture medium, various numbers (Di, Tri, Tetra and Hexa) of juvenile sporophytes per gametophyte were formed and showed 1.03, 0.66, 0.53 and 0.76 cm of sporophyte lengths, correspondingly. Conclusion: An in vitro developed polyembryony or juvenile sporophyte exhibited normal growth in their morphological structure.