Abstract: The biological effect of proton radiation is an important foundation of space radiobiology and proton radiotherapy, which can provide scientific basis for the estimation of risk to crews in space radiation and optimization design of proton therapy. It is necessary to establish irradiation technology for biological samples by accelerator. The protons with middle-energy generated by 100 MeV cyclotron established recently by the China Institute of Atomic Energy have the highest energy in China, particularly suitable for the research of space radiobiology and proton therapy. In present study, the methods of proton beam diagnosis and dose measurement were established. The beam quality, such as the size and uniformity of irradiation field, and the accuracy of dose measurement system, was analyzed and evaluated by means of in-beam and off-line methods. The results show that LiF(Mg, Ti) thermo-luminescence detector with good response to photon dose also has good dose response to 90 MeV protons, which can be used to evaluate the accuracy of dose of protons with middle-energy. The uniformity of 100 MeV proton beam is better than 90% in 5.0 cm×5.0 cm irradiation field. The accuracy of in-beam dose measurement system is better than 93%. The beam quality and dose measurement conditions of proton basically meet the requirements of radiobiology, which can provide reliable guarantee for the research of radiobiological effects.