Background. To investigate the clinical significance of color ultrasound, magnetic resonance imaging (MRI), miR-21, and CA199 in the diagnosis of pancreatic cancer (PC). Methods. A total of 160 patients with PC admitted to our hospital from April 2018 to February 2021 were included in the PC group, and another 100 patients with benign pancreatic disease during the same period were included in the pancreatic benign disease group. Color ultrasound and MRI were used for imaging examination of the two groups of PC patients, and the sensitivity, accuracy, and specificity of the two methods for preoperative diagnosis of PC were calculated, respectively. A total of 100 healthy people who underwent physical examination during the same period were included in the control group. Serum CA199 levels of the three groups were detected by ELISA assay. The level of serum miR-21 in the three groups was detected by qRT-PCR. A receiver operating curve (ROC) was drawn to analyze and calculate the sensitivity, specificity, and accuracy of the two serum markers and the combination of color ultrasound and MRI in the detection of PC. Results. Serum CA199 and miR-21 levels in the PC group were significantly higher than those in the benign lesion group and control group. CA199 and miR-21 levels in the benign lesion group were higher than those in the control group. Both color ultrasound and MRI showed a higher detection rate for PC, and the sensitivity and accuracy were significantly higher than those of CA199 and miR-21. The sensitivity, specificity, and accuracy of combined detection were 91.88%, 96.00%, and 93.46%, respectively, which were significantly higher than those of single detection. Conclusion. The combined detection of color ultrasound, MRI, miR-21, and CA199 have a high application value in the early diagnosis of PC, which can effectively improve the sensitivity and accuracy of clinical diagnosis, reduce the probability of missed diagnosis and misdiagnosis, and provide a reference for the rational clinical treatment plan and prognosis.