Nano-selenium is biosynthesized using lemon extract as a raw material. The formation process of nano-selenium is tracked by UV-Vis technology, the chemical composition and structure of the synthesized nano-selenium are characterized by XRD, the surface morphology is observed by SEM, and the nano-particle size is determined by a particle size analyzer. FTIR was used to analyze the chemical interaction between the nano-selenium surface and biomolecules. The scavenging abilities of the prepared nano-seleniumon on superoxide anion free radicals, hydroxyl free radicals and DPPH free radicals were examined. It was found that at pH = 10, the lemon extract can reduce 50 mmol / L selenite to produce amorphous selenium nanoparticles. The particle size of the synthesized nano-selenium is between 78.8~122.4 nm at 1 h. At 2 h, the particle size of nano-selenium is between 164.2~295.3 nm, which is spherical and has good dispersion; the lemon water extract is used both as a reducing agent and as a stabilizer for synthesizing nano-selenium. The essential reason is the chemical action between the biomolecule and the nano-selenium surface. Nano-selenium shows antioxidant activity positively correlated with concentration. Comparative studies show that the antioxidant strength is in order of vitamin C> small-sized nano-selenium> large-sized nano-selenium.