Currently, the advancement in non-thermal atmospheric plasma technology enables plasma treatments on some heat-sensitive targets, including biological substances, without unspecific damage caused by thermal effect. The significant effects of non-thermal atmospheric plasma modulating biological events have been demonstrated by considerable studies. Protein, one of the most important biomolecules, participates in the majority of the life-sustaining activities in all organisms, whose functions are derived from the diverse biochemical properties of amino acid compositions and four-tiered protein structure hierarchy. Therefore, the knowledge of how non-thermal atmospheric plasma affects protein greatly benefits the understanding and application of the non-thermal atmospheric plasma’s effect in biological area. In this review, we summarize recent research progress on the effects of non-thermal atmospheric plasma, particularly its reactive species, on biochemical and biophysical characteristics of proteins at different structural levels that leads to their functional changes. Moreover, the physiological effects of non-thermal atmospheric plasma at cellular or organism level driven by the manipulations on protein and their relative application prospects are reviewed. Despite the exceptional application potential, the exploration of the non-thermal atmospheric plasma’s effect on protein still confronts with difficulties due to the limited knowledge of the underlying mechanisms and the complexity of non-thermal atmospheric plasma operation systems, which requires further studies and standardization of non-thermal atmospheric plasma treatments.