Burn injury is highly traumatic for paediatric patients, with the severity of the burn often dictating the extent of scar formation. The diagnosis of burn wound severity is largely determined by the attending clinician's experience. Thus, a greater understanding of the biochemistry at burn wound site environment and the biology of burns of different severities at an earlier stage may reduce the reliance on subjective diagnoses. In this study, blister fluid was collected from superficial thickness, deep-partial thickness, and full-thickness paediatric burn wounds. Samples were combined together based on burn depth classification and then subjected to four different fractionation methods followed by trypsin digestion. Peptides were analysed by liquid chromatography tandem mass spectrometry in order to measure the proteome of each fraction. In total, 811 individual proteins were identified, including 107, 84, and 146 proteins unique to superficial, deep-partial thickness and full-thickness burn wounds, respectively. The differences in the protein inventory and the associated gene ontologies represented within each burn depth category demonstrated that there are subtle, yet significant, variations in the biochemistry of burn wounds according to severity. Importantly, this study has produced the most comprehensive catalogue of proteins from the paediatric burn wound microenvironment to date.To our knowledge, this study has been the first to comprehensively measure the paediatric burn blister fluid proteome and has provided insight into the proteomic response to burn injury. The study contributes to the knowledge of blister fluid biochemistry of burn injury and provides clinically relevant knowledge through the qualitative evaluation of biochemical differences between burns of different depths. A better understanding of the burn wound environment will ultimately assist with more accurate clinical decision making and improved wound healing and scar reduction procedures.