We conducted a pooled analysis comparing the efficacy of an immunohistochemistry (IHC) assay using the D5F3 antibody with that of fluorescence in situ hybridization (FISH) for detecting ALK gene rearrangement in NSCLC patients. A total of 32 studies involving 5805 samples were included in this review. Pooled sensitivity for D5F3 IHC was 0.97 (95% CI: 0.93-0.98), specificity was 0.99 (95% CI: 0.98-1.00), PLR was 119.20 (95% CI: 57.79-245.89), NLR was 0.03 (95% CI: 0.02-0.07), DOR was 3526.66 (95% CI: 1344.71-9249.03), and AUROC was 1.00 (95% CI: 0.99-1.00). Meta-regression revealed that specimen type was a source of heterogeneity for specificity, and specimen type and FISH signal distance were sources of heterogeneity in the joint model. Subgroup analysis revealed that sensitivity and specificity were higher when the FISH signal distance standard was >= 2 than when it was >= 1. Sensitivity was higher for tumor specimens than for cell specimens; specificity was higher for cell specimens than for tumor specimens. In conclusion, the D5F3 IHC assay was nearly as effective as FISH for detection of ALK gene rearrangement in NSCLC patients.