Objective To investigate the expression and mechanism of TUBB3 in pediatric medulloblastoma. Methods The expression of TUBB3, PD-L1 and tumor stem cell markers in medulloblastoma of children were detected by immunohistochemistry and their correlation was analyzed. The effect of TUBB3 on the proliferation and invasion of medulloblastoma cells was detected by CCK-8 and TRANSWELL. The effect of p53 expression on TUBB3 expression was analyzed by Western blotting. Results The positive rate of TUBB3 was 82.1%. TUBB3 expression is related to the degree of tissue differentiation, lymph node metastasis and estrogen receptor level. Kaplan-Meier analysis showed that high expression of TUBB3 was associated with low overall survival after medulloblastoma surgery. Knocking down the expression of TUBB3 inhibited the proliferation and invasion of Daoy and D341 cells. TUBB3 was positively correlated with the expression of CD44, CD133, PD-L1 and p53 in pediatric medulloblastoma. WB showed that TUBB3 expression was up-regulated by p53 gene knockdown. Conclusion TUBB3 plays a carcinogenic role in pediatric medulloblastoma and is a potential biomarker and therapeutic target for pediatric medulloblastoma.