Rooting of orchids is still the main real problem that obstructs its propagation, as roots are hardly to develop in nature. So, Orchid needs to improve its propagation by enhancing its rooting development in vitro. The plant tissue culture of orchids is the most promising field that has been commercially used for getting of virus-free and high-quality rooted plants and representing around 70 % of transplants production. In this study the, shoots of in vitro plantlets were separated and cultured on half MS medium supplemented with nano-Se (0,10, 20, 30, 40 and 50 mg l-1) or corresponding doses of bulk type selenite (0, 0.5, 1, 2, 4, 6, 8 and 10 mg l-1) in separates treatments with or without 0.5 mg l-1 NAA (α-naphthalene acetic acid). The two different sources of selenium (i.e., nano-Se and sodium selenite) were selected at mentioned concentrations to improve orchid rooting and acclimatization. Supplementation of half MS medium with 50 mg l-1 nano-Se + 0.5 mg l-1 NAA recorded the highest significant value of rooting percentage and root length as well. Whereas, half MS medium with 40 mg l-1 nano-Se + 0.5 mg l-1 NAA gave the optimum number of roots. On the other hand, this enhancing effect decreased after fortified the culture medium with 10 mg l-1 selenite. It could be concluded that in vitro rooting of Phalaenopsis orchid was improved by supplementation of nano-Se up to 50 mg l-1 in presence of 0.5 mg l-1 NAA consequence, acclimatization process was enhanced.