The seasonal changes in the chemical composition of Olea europaea leaves from January to December at Liangshan (China) have been investigated. The highest total phenolic content (TPC), total flavonoid content (TFC), and free amino acid content (FAAC) levels were found in May and December, while the lowest levels were detected in April and September. The soluble protein content (SPC) and the soluble sugar content (SSC) were highest in spring but lowest in summer and winter. The levels of major phenolic compounds, including oleuropein, and luteolin-4’-O-glucoside, followed by apigenin-7-O-glucoside, quercetin, rutin, luteolin, and apigenin, increased during spring and winter but decreased during summer and autumn. In addition, phenolic extracts (PEs) showed dose-dependent antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and superoxide radical scavenging activity assays; the reducing power was tested. The anticancer activities of PE at various concentrations were assessed by a cell counting kit-8 (CCK-8), and the IC50 (50% effective concentration) to HEK293, HeLa, and S180 cells were 841.48, 7139, and 457.69 μg/mL, respectively. PE-treated S180 cells inhibited proliferation through activation of caspase-3/9 and disruption of the mitochondrial membrane potential. Thus, PE in Liangshan olive leaves possessed strong antioxidant and anticancer potential, and spring and winter were determined as optimal harvesting seasons.