Objective To investigate the expression levels and significance of matrix metalloproteinase-9 (MMP-9) and hypoxia-inducible factor-1α (HIF-1α) in the serum of patients with acute ischemic stroke at high altitude. Methods A total of 60 patients with acute ischemic stroke in high altitude who were admitted to our hospital from September 1, 2017 to November 1, 2019 were enrolled as observation group, and 60 healthy individuals were enrolled as control group. ELISA was used to measure the expression of MMP-9 and HIF-1α in serum, and the association of these two factors with the severity and prognosis of acute ischemic stroke was analyzed. Results The observation group had significantly higher expression of MMP-9 and HIF-1α in serum than the control group (both P=0.000). There were significant differences in the expression of MMP-9 and HIF-1α in serum between the patients with different degrees of injury, infarct areas, and prognoses (P<0.05). The high expression levels of MMP-9 and HIF-1α were independent risk factors for poor prognosis (P<0.05). Conclusions There are significant increases in the expression levels of MMP-9 and HIF-1α in serum in patients with acute ischemic stroke at high altitude, which are associated with the degree of brain injury, cerebral infarct area, and prognosis, and therefore, they may have a certain value in evaluating the prognosis of patients.