Background Emerging evidence has shown the damage of air pollution and the benefits of physical activity to human health, and the effects of air pollution and physical activity on the nervous system need more research. Objective To explore the effects of short-term air pollution exposure and physical activity on neural damage biomarkers in healthy elderly. Methods Using a design of panel study, physically and mentally healthy retired employees were recruited from Xinxiang Medical University, and were followed up five times regularly from December 2018 to April 2019. The demographic characteristics and physical activity information were obtained by questionnaire, and the weekly physical activity level was calculated according to intensity and duration of physical activity. Biomarkers of neural damage in serum were measured, including brain-derived neurotrophic factor (BDNF), neurofilament light chain (NF-L), neuron specific enolase (NSE), protein gene product 9.5 (PGP9.5), and S100 calcium-binding protein B (S100B). Air pollution data (including PM_2.5, PM₁₀, O₃, SO₂, CO, and NO₂) of the follow-up period were collected. Generalized estimation equation was used to analyze the association of air pollution concentration and physical activity level with the concentration of neural damage biomarkers. Results A total of 29 volunteers were included in the study, with an average age of (63.5±5.9) years; there were 11 men accounting for 37.93%; more than half of them (62.07%) received above junior middle school education; the mean physical activity level was (80.23±54.51) MET-h·week⁻¹. The daily average concentrations of PM_2.5, PM₁₀, O₃, SO₂, CO, and NO₂ during the study period were (68.27±60.98) μg·m⁻³, (130.57±58.71) μg·m⁻³, (36.86±13.89) μg·m⁻³, (17.86±10.59) μg·m⁻³, (4.94±1.34) mg·m⁻³, and (50.83±8.03) μg·m⁻³, respectively. The average serum concentrations of BDNF, NF-L, NSE, PGP9.5, and S100B were (139.12±46.71) μg·L⁻¹, (402.60±183.31) ng·L⁻¹, (11.26±10.32) ng·L⁻¹, (14.32±13.57) ng·L⁻¹, and (127.57±41.74) ng·L⁻¹, respectively. The results of generalized estimation equation showed that a higher concentration of PM_2.5 or O₃ was associated with increased serum NSE (OR=1.359, 95%CI: 1.224-1.509, P<0.001; OR=1.286, 95%CI: 1.076-1.537, P=0.006), while a higher concentration of NO₂ was associated with decreased serum NSE (OR=0.692, 95%CI: 0.549-0.873, P=0.002); a higher concentration of O₃ or SO₂ was related to the reduction of serum NF-L concentration (OR=0.855, 95%CI: 0.740-0.989, P=0.035; OR=0.813, 95%CI: 0.700-0.946, P=0.007); a higher concentration of NO₂ was associated with decreased PGP9.5 in serum (OR=0.866, 95%CI: 0.777-0.965, P=0.009); a higher level of physical activity was associated with increased serum S100B (OR=1.038, 95%CI: 1.003-1.074, P=0.034); and no significant association of physical activity level or air pollution with BDNF (P>0.05). Conclusion Acute exposure to air pollution and high-level physical activity might affect the neural damage of elderly populations. Specifically, particulate matter (PM_2.5) could increase NSE, while gaseous pollutants (O₃, NO₂, and SO₂) could decrease NF-L and PGP9.5.