Biodiesel production from oleaginous microalgae shows great potential as a promising alternative to conventional fossil fuels. Currently, most research focus on algal biomass production with autotrophic cultivation, but this cultivation strategy induces low biomass concentration and it is difficult to be used in large-scale algal biomass production. By contrast, heterotrophic algae allows higher growth rate and can accumulate higher lipid. However, the fast-growing and lipid-rich microalgae that can be cultivated in heterotrophic system for the industrial application of biodiesel production are still few. Traditional solvent extraction and gravimetric determination to detect the microalgal total lipid content is time-consuming and laborious, which has become a major limiting factor for selecting large number of algae specimens. Thus, it is critical to develop a rapid and efficient procedure for the screening of lipid-rich microalgae. A novel green microalga Scenedesmus sp. strain R-16 with high total lipid content was selected using the Nile red staining from eighty-eight isolates. Various carbon sources (fructose, glucose and acetate) and nitrogen sources (nitrate, urea, peptone and yeast extract) can be utilized for microalgal growth and lipid production, and the optimal carbon and nitrogen sources were glucose (10 g L^-1) and nitrate (0.6 g L^-1), respectively. Compared to autotrophic situation, the strain R-16 can grow well heterotrophically without light and the accumulated total lipid content and biomass reached 43.4% and 3.46 g L^-1, respectively. In addition, nitrogen deficiency led to an accumulation of lipid and the total lipid content was as high as 52.6%, and it was worth noting that strain R-16 exhibited strong tolerance to high glucose (up to 100 g L^-1) and a wide range of pH (4.0-11.0). The newly developed ultrasonic-assisted Nile red method proved to be an efficient isolation procedure and was successfully used in the selection of oleaginous microalgae. The isolated novel green microalgal strain R-16 was rich in lipid and can live in varied and contrasting conditions. The algae appeared to have great potential for application in microalgae-based biodiesel production.