In this paper, energy loss of alpha particles in different vacuum levels is studied experimentally and via theoretical analysis. A better understanding of energy loss of α particle in vacuum will help detect more exact numbers of alpha particles. Two ²³⁹Pu sources are used to measure the energy loss of α particle crossing different vacuum levels (different air pressures). The experimental data are obtained from an instrument – PAM-100 developed by authors. The experimental results have shown that increasing vacuum levels will lead to more alpha residual energy but less energy loss. When the vacuum level reaches 0.04 MPa, alpha particles (²³⁹Pu, 5.115 MeV) will lose the energy of about 0.175 MeV with traversing 5 mm distance. Theoretical calculations have shown a good agreement with experimental results. This implies that the instrument has a high accuracy and could be applied in field work.