Pseudomonas, due to its diversity in habitat and metabolic type, makes it have broad prospects applying in bioremediation, bioconversion, and biocontrol, while the introduction of exogenous gene is the key link to genetically modified Pseudomonas. The preparation and transformation of competent cells are the important methodological basis of the introduction of exogenous gene. In this paper, three Pseudomonas strains (P. putida TS11, P. stutzeri DNB, and P. mendocina JJ12) isolated from a petroleum-contaminated soil were taken as the recipient strains, and a three-factor and four-level orthogonal experiment was conducted to investigate the effects of CaCl₂ concentration, heat shock duration, and recovery duration on the preparation and transformation efficiency of the strains competent cells. The results showed that CaCl₂ concentration was the most important factor affecting the transformation efficiency (P<0.05), and the transformation efficiency was improved markedly when the Pseudomonas cells were repeatedly washed with sterile distilled water before the preparation of competent cells. When the P. putida TS11 cells were treated with 100 mmol·L^-1 of CaCl₂, heatshocked for 3 minutes at 42 ℃, and incubated for 1.5 hours at 30 ℃, the P. stutzeri DNB cells were treated with 50 mmol·L^-1 of CaCl_2, heatshocked for 6 minutes, and incubated for 1.5 hours, and the P. mendocina JJ12 cells were treated with 75 mmol·L^-1 of CaCl₂, heat shocked for 4.5 minutes, and incubated for 0.5 hours, the transformation efficiency of exogenous plasmids in the three strains all achieved 10.5 cells·μg^-1 DNA.