The invention belongs to the fields of petroleum biotechnology and environment biotechnology, and concretely relates to a preparation method of a Pseudomonoas aeruginosa inoculant and a rhamnolipid broth. The method comprises the following steps: inoculating 2-8V/V% of an expansion-cultured Pseudomonoas aeruginosa seed liquid into a disinfected culture medium of a fermentation tank, adjusting the pH value of the fermentation culture medium, ventilating, and carrying out stirring fermentation to obtain the Pseudomonoas aeruginosa inoculant or the rhamnolipid broth, wherein the fermentation culture medium contains essential carbon source, nitrogen source, inorganic salt and trace elements, The Pseudomonoas aeruginosa inoculant and the rhamnolipid broth prepared through the method can well adapt to oil reservoir environment, and also have good oil dispelling effect.