Objective To investigate the expression levels of signal transduction molecule 3 (SMAD3) and white blood cell differentiation antigen 44 (CD44) in papillary thyroid carcinoma (PTC) tissue and their relationship with clinicopathological features and prognosis. Methods A total of 88 PTC patients admitted to our hospital from June 2019 to June 2020 were selected as the study objects. The carcinoma tissue and adjacent tissue (more than 3 cm away from the carcinoma tissue) were collected and included into the PTC group and adjacent group. Real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of SMAD3 and CD44, and the positive expressions of SMAD3 and CD44 were detected by immunohistochemistry. Spearman correlation analysis was performed to determine the mRNA expression levels of SMAD3 and CD44 in the carcinoma tissue of PTC patients. The relationship between SMAD3 and CD44 and the prognosis of PTC patients was analyzed using the Kaplan-Meier method. COX regression was applied to analyze risk factors affecting the prognosis of PTC patients. Results Compared with the adjacent group, the expression level of SMAD3 in the PTC group was obviously reduced, while the expression level of CD44 was obviously increased (t=8.484, 11.232, P<0.05). Correlation analysis showed that the mRNA expression levels of SMAD3 and CD44 in the carcinoma tissue were negatively correlated (r_s=-0.519, P<0.05). Compared with the adjacent group, the positive expression rate of SMAD3 in the PTC group was obviously reduced, while the positive expression rate of CD44 was obviously increased (χ²=41.905, 62.888, P<0.05). The expression of SMAD3 was associated with TNM stage (χ²=7.678,P=0.006), lymph node metastasis (χ²=20.398, P<0.05) and invasion of envelope (χ²=9.881,P=0.002). The expression of CD44 was associated with TNM stage (χ²=3.959,P=0.047), lymph node metastasis (χ²=20.702,P<0.05) and invasion of envelope (χ²=10.363,P=0.001). After 3 years of follow-up, the survival rate of PTC patients with SMAD3 positive expression was higher than that of the patients with SMAD3 negative expression (χ²=4.644,P=0.031). The survival rate of PTC patients with CD44 positive expression was significantly lower than that of those with negative expression (χ²=5.331,P=0.021). Multivariate COX regression analysis showed that lymph node metastasis, SMAD3 and CD44 expression were risk factors affecting the prognosis of PTC patients (P<0.05). Conclusion The expression of SMAD3 is abnormally low and the expression of CD44 is high in PTC tissue, which is related to the clinicopathological characteristics and prognosis of PTC patients.