Protein is an essential component of organisms. Damages and diseases of the body could be known from analysis of proper proteins， and thus analysis of proteins by capillary electrophoresis（CE） is important in the field of clinical medicine. However， the current researches on pulsed field capillary electrophoresis（PFCE） mainly focus on the improvement of separation performance of DNA， and there is little research about protein analysis by PFCE. To improve the separation performance of proteins， lysozyme， growth hormone， carbonic anhydrase， actin， bovine serum albumin and phosphorylase B were taken as the samples， using hydroxyethyl cellulose（HEC） as the sieving polymer， applying the capillary with 6 cm of effective length and 8 cm of total length， performing the PFCE of proteins， and investigating the effect of pulse frequencies and modulation depth on the separation performance of proteins sized range of 14.4－97.4 kDa for the first time. Results demonstrated that， compared with the direct current CE（DCCE） under 100 V/cm of electric field strength， the PFCE employing 1.4% HEC as the sieving polymer， a 100 V/cm of average electric field strength， a 10 Hz of pulsed frequency and a 250% of modulation depth would make the migration time of proteins with same molecular weight decrease about 6.6%－9.6%. The resonant frequency for the protein and pulsed electric field was 10 Hz. The migration time of protein molecule was increased with the growth of pulsed frequency when the pulsed frequency was lower than the resonant frequency. In contrast， the migration time was decreased if the pulsed frequency was higher than the resonant frequency. The resolution could be increased about 34.1%－88.1%， and the maximum theoretical plate number reached to 4.03 × 10,4, if the frequency of the pulsed electric field was the same as the resonant frequency. Under the same average electric field strength， the migration time of protein decreased with the growth of modulation depth from 0 to 250%， and the Joule heating in the sieving polymer induced by pulsed electric field increased 2.64 times than that under direct current electric field. The growth of Joule heating will make the viscosity of the polymer reducing. Consequently， the decrease of the migration time was more obvious if the molecular weight of protein was larger. The migration time of phosphorylase B was reduced about 9.6% when the modulation depth was raised from 0 to 250%. The resolution between the adjacent protein molecules was not obviously deteriorated， even though the migration time was shorter. This work provides a new method for improving the separation performance of protein.