Purpose Azacitidine (AZA), a hypomethylating agent, is the standard treatment for patients with myelodysplastic syndromes (MDS). However, responses are generally transient in approximately 50% of patients. Cytomorphology and immunophenotyping are widely used in the treatment of MDS. Therefore, the present study investigated the relationship between immunophenotyping and the outcomes of MDS patients administered AZA as first-line therapy. Methods We retrospectively examined the relationship between immunophenotyping and the outcomes of 118 MDS patients administered AZA as first-line therapy. Results The response to AZA was poorer in patients with a CD33/CD34 ratio > 1 than in those with a CD33/CD34 ratio < 1. Univariate and multivariate analyses identified higher serum LDH levels, higher risk IPSS-R, and a CD33/CD34 ratio > 1 as independent adverse prognostic factors for overall survival. A Kaplan-Meier analysis showed that a CD33/CD34 ratio > 1 was associated with poor survival. Conclusion In the present study, a CD33/CD34 ratio > 1 in blast cells was associated with poor survival in MDS patients administered AZA as first-line therapy.