Pseudomonas, an important spoilage bacterium in animal-derived foods as well as fruits and vegetables in the cold chain, can cause huge economic losses to the food industry. The food spoilage ability of Pseudomonas is closely related to the formation of biofilm, which can enhance the survival and adaptability to the food processing and storage environment of Pseudomonas, causing persistent contamination. However, a systematic review of the biofilm formation of Pseudomonas and its mechanism is still lacking at present. This article describes the contamination status of Pseudomonas in fresh foods, analyzes the biofilm formation and extracellular matrix composition of Pseudomonas, and elucidates the major regulatory factors of the biofilm formation of Pseudomonas such as cyclic diguanylate, quorum sensing system and small RNAs. We believe that this review will provide a theoretical reference for uncovering the mechanism of food spoilage caused by Pseudomona and controlling its biofilm contamination.