In this study, a total of 45 female Beijing-You chicken (BYC) hens, fed on the same diet, were collected at the slaughter age of 150, 300, or 450 days (D150, D300, and D450) from sexual maturation stage to culling stage (15 birds per age). Gas chromatography-mass spectrometry (GC-MS) analysis strategies and tandem mass tag (TMT)-LC-MS/MS proteomics were applied for profiling fatty acid (FA) compositions and changes in the related metabolisms based on analyses of the differentially expressed proteins (DEPs), respectively, in hen breast meat between the slaughter ages. The FA profiling showed that increasing hen ages resulted in increased contents of both saturated and unsaturated fatty acids. Proteomic analyses showed a total of 4935 proteins in chicken breast muscle with the false discovery rate (FDR) < 1% and 664 of them were differentially expressed (fold change > 1.50 or < 0.67 and P < 0.01). There were 410 up- and 116 down-regulated proteins in D150 vs. D300 group, 32 up- and 20 down-regulated in D150 vs. D450 group, and 72 up- and 241 down-regulated in D300 vs. D450 group. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that 57 DEPs were related to FA/lipid-related metabolisms. These DEPs presented 21 significantly enriched (P < 0.05) pathways, including well-known pathways for FA metabolism, desaturation, and elongation, and the signaling pathways for lipid metabolism (PPAR, adipocytokine, calcium, VEGF, MAPK, and Wnt). In addition, there existed several representative DEPs (FABP, FABP3, apoA-Ⅰ, apoA-IV, apoC-Ⅲ, apoB, VTG1, and VTG2) involved in the regulation of FA/lipid transportation. These results indicated that FA metabolisms were altered at different levels in hen meat during the laying. The construction of the interaction networks revealed that HADH, ACAA2, HADHA, ACSL1, CD36, CPT1A, PPP3R1, and SPHK1 were the key core nodes. Finally, eight DEPs were quantified using parallel reaction monitoring (PRM) to validate the results from TMT analysis. Overall, this study expands our understanding of how the laying age affects the FA compositions and metabolism in hen meat. The data might help provide the strategies in the future to control the FA compositions in chicken meat.