Background The production of biopolymers from waste resources is a growing trend, especially in high-population countries like Egypt. Beta-glucan (β-glucan) belongs to natural polysaccharides that are derived from plant and microbial origins. In this study, following increasing demands for β-glucan owing to its bioactive properties, a statistical model to enhance microbial β-glucan production was evaluated for its usefulness to the food and pharmaceutical industries. In addition, a trial to convert β-glucan polymer to nanostructure form was done to increase its bioactivity. Results Ingredients of low-cost media based on agro-industrial wastes were described using Plackett–Burman and central composite design of response surface methodology for optimizing yeast β-glucan. Minerals and vitamin concentrations significantly influenced β-glucan yield for Kluyveromyces lactis and nitrogen and phosphate sources for Meyerozyma guilliermondii. The maximum predicted yields of β-glucan recovered from K. lactis and M. guilliermondii after optimizing the medium ingredients were 407 and 1188 mg/100 ml; respectively. For the first time, yeast β-glucan nanoparticles (βGN) were synthesized from the β-glucan polymer using N-dimethylformamide as a stabilizer and characterized using UV–vis spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FT-IR). The average size of βGN was about 300 nm as determined by DLS. The quantitative variation of functional groups between β-glucan polymer and βGN was evaluated by FT-IR for explaining the difference in their biological activity against Normal Homo sapiens-Hela contaminant and Hepatic cancer cell lines. Conclusions Enriching the low-cost media based on agro-industrial wastes with nutritional ingredients improves the yield of yeast β-glucan. The present study succeeds to form β-glucan nanoparticles by a simple method.