Objective To investigate and analyze the distribution of serum cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and squamous cell carcinoma-associated antigen (SCCA) levels in healthy pregnant women during pregnancy and to assess their diagnostic value for cervical cancer in pregnancy. Methods A total of 441 healthy pregnant women and 69 patients with cervical cancer in pregnancy who attended the Obstetrics and Gynecology Hospital of Fudan University from Jan 2021 to May 2024 were selected, and 165 healthy women in the Physical Examination Center of the Obstetrics and Gynecology Hospital of Fudan University were included in the same period as the control group. The healthy pregnant women were divided into 143 in early pregnancy (T1 group), 147 in middle pregnancy (T2) and 151 in late pregnancy (T3). Serum CYFRA21-1 and SCCA values were detected and analyzed in all groups. Oneway ANOVA, independent samples t-test, Mann-Whitney U-test, Kruskal-Wallis H-test, logistic analysis, and ROC curves were used for comparative analysis. Results The CYFRA21-1 and SCCA values were 1.66 (1.19-2.17) ng/mL and 0.8 (0.6-1.0) ng/mL in the control group, 3.07 (2.11-4.14) ng/mL and 0.9 (0.7-1.3) ng/mL in the healthy pregnant women group, and were 4.33 (2.99-7.60) ng/mL and 1.8 (0.9-8.5) ng/mL in the patients with cervical cancer in pregnancy group, respectively. There was a statistically significant difference in the two serum values between every two groups (P<0.05). CYFRA21- 1 levels were 3.13 (2.46-4.05) ng/mL, 1.89 (1.50-2.53) ng/mL and 4.19 (3.48-5.43) ng/mL in the T1, T2, and T3 groups, respectively; and SCCA levels were 0.9 (0.7-1.1) ng/mL, 0.7 (0.6-1.0) ng/mL and 1.2 (0.8-1.7) ng/mL, respectively. The results of T1 and T3 groups were higher than those of the control group (P<0.05); however, there was no statistically significant difference between the results of the T2 group and those of the control group (P>0.05). The areas under the ROC curves for the diagnosis of cervical cancer in pregnancy for CYFRA21-1, SCCA, human epididymis protein 4 (HE4), anticarcinoembryonic antigen (CEA) and joint indicators were 0.684, 0.724, 0.612, 0.791 and 0.913, with sensitivities of 36%,48%,38%,57% and 73%,specificities of 96%,97%,89%,86% and 99%, respectively. The cut-off values of each indicator were 6.05 ng/mL, 2.60 ng/mL, 51.45 pg/mL and 1.75 ng/mL, respectively. Conclusion Serum CYFRA21-1 and SCCA levels were higher in pregnant women during early and late pregnancy compared with non-pregnant individuals, while they were not statistically different from non-pregnant women during mid-trimester. CYFRA21-1 and SCCA have diagnostic value for patients with cervical cancer during pregnancy. [ABSTRACT FROM AUTHOR]