Purpose <p>Conventional spray drying often produces heterogeneous enzyme powders because of broad droplet size distributions and complex turbulent flow fields, limiting mechanistic understanding of particle formation and quantitative structure–activity analysis. This study aimed to fabricate uniform lysozyme microspheres and clarify how formulation and drying conditions govern their microstructure and enzymatic activity.</p> Methods <p>A micro-fluidic jet spray dryer was used to prepare highly uniform pure lysozyme and excipient-containing lysozyme microspheres. The effects of carbohydrate excipients and drying temperature on particle morphology, secondary structure, surface composition, and enzymatic activity were systematically evaluated.</p> Results <p>Pure spray-dried lysozyme microspheres showed a highly uniform single-hole bowl-like morphology with smooth surfaces. However, drying-induced thermal and shear stresses reduced α-helix content and caused activity loss. Carbohydrate excipients altered the structural evolution pathways. Surface elemental analysis indicated lysozyme enrichment in most formulations, except those containing dextran T40. Mannitol crystallization induced phase separation, lysozyme unfolding, and inactivation. Trehalose preserved conformational stability through a hydrogen-bonded glassy matrix, whereas dextran T40 mainly suppressed lysozyme aggregation through steric hindrance.</p> Conclusions <p>This work establishes a formulation properties/process–microstructure–enzymatic activity relationship in a uniform microsphere system, supporting the rational design of performance-predictable spray-dried enzyme powders.</p>