In this paper, ultra-high pressure micro-jet was used to separate and extract β-glucan from Euglena (EBG). The effects of microfluidizing time, microfluidizing pressure, solid-liquid ratio and sodium dodecyl sulfate (SDS) concentration on the extraction rate of EBG were studied by single factor experiment and response surface optimization design experiment, and its structure was characterized by Congo red experiment, FT-IR, XRD and SEM. The results showed that the optimum extraction conditions of β-glucan from Euglena were as follows: microfluidization time of 93 s, microfluidization pressure of 340 bar, solid-liquid ratio of 1∶270 g/mL and SDS concentration of 4.2 g/L. Under these conditions, the extraction rate of EBG was 80.27%, which was close to the theoretical value of 81.69%. Congo red experiment, FT-IR, XRD and SEM results show that EBG has an orderly triple helix structure; the crystallinity of EBG decreases; SEM test results show that cracks and voids appear on the surface of EBG. By measuring the DPPH? scavenging capacity of Euglena β-glucan standard (EBGS) and EBG, the results showed that the DPPH? scavenging rate of EBGS and EBG was basically the same at the concentration of 0.4~2.0 mg/mL, and the DPPH? scavenging rate of EBG reached 50.26% at the concentration of 4.0 mg/mL, which was higher than that of EBGS by 35.14%.