BACKGROUND: Cortisol is a steroid hormone secreted mainly by the adrenal cortex and is associated with chronic stress levels in the body. Hair cortisol concentration (HCC) is a reliable index to assess human stress levels. So far, no study has reported whether COVID-19 vaccination is associated with the changes of HCC. METHODS: Hair samples were collected from 114 college students at Hangzhou City University and Zhejiang University. Among them, 57 cases completed COVID-19 vaccination and others did not. HCCs were measured by the chemiluminescence immunoassay (CLIA). The psychological stress levels were evaluated using the Chinese College Student Psychological Stress Scale (CCSPSS). General information and adverse reactions of the subjects were collected by questionnaire. RESULTS: Compared with the vaccinated college students, the unvaccinated students had higher HCC levels in both A and B hair segments respectively corresponding older or six weeks before and newer or six weeks after vaccination (p < 0.05), reflecting higher stress levels. Besides, the vaccinated group had significantly higher HCCs in segment B compared with segment A (p < 0.05). Further analysis showed that the value of ΔHCC (HCCseg.B - HCCseg.A) of the vaccinated group was strongly associated with COVID-19 vaccination (p < 0.05), but was not associated with age, gender, BMI, CCSPSS score, hormone use, exercise frequency, hair washing frequency, or hair treatment. Finally, the number of self-reported systemic adverse reactions in the vaccinated group was associated with ΔHCC (p < 0.01). CONCLUSION: The COVID-19 vaccination had an impact on the value of HCC, which might be linked to the occurrence of systemic adverse effects following vaccinations.