Objectives The tear, as an important bodily secretion, plays a crucial role in preventing infection and maintaining homeostasis of ocular surfaces. Although accumulating studies have reported on the HIV-1 viral load profile among varying bodily fluids and secretions, little was known concerning HIV-1 dynamics in tears. Therefore, the objectives of this study were to investigate the HIV-1 viral load in tears of HIV/AIDS patients and study factors influencing their tear viral load. Methods A cross-sectional study was conducted. 67 patients with a confirmed HIV-1 infection or AIDS were recruited from the Beijing You'an Hospital, China between April 2018 and September 2018. Socio-demographic information and laboratory test results were collected. At the same time, ophthalmic examinations were carried out and tear samples were tested. Results Of 30 highly active antiretroviral therapy (HAART)-naive patients, 53.3% had detectable HIV-1 RNA in tears. Of 37 patients on HAART, HIV-1 RNA was undetectable in their tears, regardless of treatment duration and blood viral load. Tear viral load ranged from TND (target not detected) to 13,096 copies/mL. Viral load was lower in tears than in blood plasma (p < 0.001), and was significantly correlated with plasma viral load (Rho = 0.566,p < 0.001) and AIDS stage (Rho = 0.312,p = 0.01), but negatively correlated with CD4 (+) T cell count, CD4 (+)/CD8 (+) T cell count, and duration of HIV infection (Rho = -0.450, Rho = - 0.464, Rho = - 0.565;p < 0.001). Conclusions HIV-1 RNA is present in tears of more than half of the HAART-naive patients, whereas absent in tears of patients on HAART. Tear viral load is positively associated with plasma viral load while it is negatively correlated with CD4 cell count. This study provides novel insights into the area with limited understanding-HIV-1 viral load in tears.