Objective To study the effect of a new mucA gene mutation on the biofilm formation process and the morphology of matured biofilm of Pseudomonas aeruginosa. Methods The mucA gene of PAO1 was cloned into the Pseudomonas aeruginosa expression plasmid pUCP20. The recombinant plasmid was transformed to mucoid PA17 which contained a new mucA mutation. Positive clones of the plasmids were identified by enzyme digestion and sequencing. The expression levels of algD in the positive clones were assessed by semi-quantitative RT-PCR. The modified plate culture method was used to establish the biofilm models of PA17, PA17 with recombinant plasmid and PAO1 in vitro. Results Transformation was identified by the decreased expression of algD in positive clones. The rate of biofilm formation of the positive clones was between those of PAO1 and PA17. The irreversible adhesion occurred after 8 h and the matured biofilm was observed on day 6. The morphologies of PA17, PAO1 and PA17 with recombinant plasmid were the same. Conclusion The mucA gene mutation of PA17 delays the formation of irreversible adhesion of PA17 biofilm, but it has no effect on the morphology of matured biofilm.