The aim of the present study was to develop an optimized niosome formulation for the encapsulation of a poorly water-soluble drug by the ecological probe sonication method. Pluronic L121 and Span 60 were used as surface active agents and the optimization of the composition was made with the aid of Design of Experiment (DoE) concept. Rifampicin was used as a model drug. Concentration levels of charge inducing agent, dicetylphosphate (DCP), and Pluronic L121 were studied as variables. Prepared niosomes with varying concentrations of DCP and Pluronic L121 resulted in small sized niosomes with sizes ranging from 190 nm to 893 nm. During the four weeks stability testing, the particle sizes were reduced slightly. The formulation containing 2 mg of DCP resulted in most stable niosomes with 75.37% entrapment efficiency. All the niosomal formulations showed higher in vitro drug release rates as compared to bulk drug formulation. As a conclusion, rifampicin loaded niosomes prepared with Pluronic L121 and Span 60 resulted in stable, small sized niosomes with improved drug release profile.