Objective To analyze the expression of fractalkine (FKN), heart-type fatty acid-binding protein (H-FABP) and D-dimer (D-D) in acute pulmonary embolism and their clinical significance. Methods A total of 43 patients with acute pulmonary embolism admitted to Zhoukou First People's Hospital from October 2020 to October 2021 were selected as the study group, and 50 healthy people who underwent physical examination during the same period were selected as the control group. The levels of serum FKN, H-FABP and D-D in the two groups were measured by double antibody sandwich ELISA and immunoturbidimetry respectively, and their expression and clinical significance were analyzed. Results Compared with low-risk and medium-risk patients in control group and study group, the levels of FKN, H-FABP and D-D in high-risk patients were higher (P<0.05), FKN, H-FABP and D-D were positively correlated with the risk grade of acute pulmonary embolism. FKN and H-FABP were positively correlated in patients with acute pulmonary embolism (r=0.503, P=0.010). There was positive correlation between FKN and D-D (r=0.682, P=0.001). There was positive correlation between H-FABP and D-D (r=0.755, P=0.001). ROC curve analysis showed that FKN, H-FABP and D-D (AUC =0.651,0.692,0.740) combined (AUC =0.844) had higher diagnostic value for acute pulmonary embolism (P<0.05). Conclusion The occurrence and pathological progress of acute pulmonary embolism may be related to the abnormal expression of serum FKN, H-FABP and D-D, and the combination of the three has important significance for the early diagnosis of acute pulmonary embolism.