Here, we present the first example of studying the aging and sexual dimorphism of brains using the zebrafish (Danio rerio) model organism by mass spectrometry (MS)-based quantitative top-down proteomics (TDP). We measured the proteoforms in male and female zebrafish brains across three ages (6, 16, and 24 months) using capillary zone electrophoresis-tandem MS (CZE-MS/MS). Our research indicates that male and female zebrafish exhibit significant differences in the abundance of important proteoforms. Significant age-related changes were observed in proteoforms associated with critical neurological processes, including neurofilament assembly, dopaminergic neuron differentiation, and synaptic vesicle priming.