Objective To provide theoretical references for early screening and prevention of nasopharyngeal carcinoma (NPC) in Linyi area by analyzing the detection results of three antibodies related to Epstein-Barr (EB) virus serological markers, Rta protein IgG antibody (Rta/IgG), early antigen IgA antibody (EA/IgA), and capsid antigen IgA antibody (VCA/IgA), in the physical examination population. Methods Three antibody test results were collected from 15873 physical examination personnel in the Physical Examination Center of Linyi People’s Hospital from October 2019 to October 2022. According to the age aggregation, they were divided into ≤30 years old group, 31~40 years old group, 41~50 years old group, 51~60 years old group and ≥61 years old group. The three antibody absorbance values were compared among the groups, and statistical analysis was performed by SPSS 26.0 software. Results The positive detection rates of Rta/IgG, EA/IgA, and VCA/IgA antibodies were 10.34%, 9.96%, and 19.30%, respectively, with statistically significant differences (χ²=780.030, P<0.05). When compared in different age groups (≤ 30 years old group, 31~40 years old group, 41~50 years old group, 51~60 years old group, ≥ 61 years old group), the overall antibody positive rate increased with age. The overall positive rates of VCA/IgA (16.76%, 18.81%, 18.85%, 19.06%, 31.61%) in different age groups were higher than those of Rta/IgG (8.54%, 8.91%, 9.89%, 11.05%, 11.63%) and EA/IgA (7.42%, 8.67%, 8.87%, 9.71%, 13.64%), with statistically significant differences (χ²=18.957, 64.805, 16.222, respectively, all P<0.05). Comparisons between genders revealed differences in the positive detection rates of Rta/IgG and VCA/IgA (χ²=5.479, 48.310, respectively, P<0.05). When analyzing the positive aggregation of three antibodies, the proportions of 3 antibodies(-), 1 antibody(+), 2 antibodies(+), and 3 antibodies(+) were 67.27%, 26.38%, 5.79%, and 0.56%, respectively, with statistically significant differences (χ²=23 319.137,P<0.05). A total of 249 male and 137 female high-risk individuals were followed up, and one male was diagnosed with NPC. Conclusion The positive rate of EB virus serological testing in the physical examination population in Linyi City is related to age and gender. Risk assessment of the test results and tracking of high-risk populations are beneficial for early screening and prevention of NPC.