Abstract Background Although the high burden of anemia among pregnant women in low-resource settings like Ethiopia is well documented, evidence is scarce on the underlying causes using biochemical tests. Therefore, this study assessed the iron status and factors associated with iron deficiency (ID) using serum ferritin concentration among pregnant women in Haramaya district, eastern Ethiopia. Methods A community-based cross-sectional study was conducted among randomly selected pregnant women in Haramaya district, eastern Ethiopia. The serum ferritin (SF) concentration was measured in the National Biochemical Laboratory of Ethiopia on a fully automated Cobas e411 (German, Japan Cobas 4000 analyzer series) immunoassay analyzer using the electro-chemiluminescence (ECL) method and standard procedures. A log-binomial regression analysis identified variables associated with iron deficiency, and defined as serum ferritin concentration < 15 μg/L (per the World Health Organization recommendation in developing countries). An adjusted risk ratio (aRR), and a 95% confidence interval (CI), were used to report associations. Finally, the p-value < 0.05 was the cut-off point for the significant association. Results A total of 446 pregnant women with a mean age of 24.78 (+ 5.20) were included in the study. A total of 236 (52.91%; 95% CI: 48.16–57.63) had iron deficiency. The overall prevalence of anemia and iron deficiency anemia (IDA) was 45.96% (95% CI: 41.32–50.71) and 28.03% (95% CI: 21.27–32.44), respectively. The risk of iron deficiency was more likely among women with low dietary diversity (aRR = 1.36; 95% CI = 1.07–1.72) and those who skipped meals (aRR = 1.29; 95% CI = 1.05–1.57), but less among women who had antenatal care (aRR = 0.73 (95% CI = 0.61–0.88). Conclusion More than half of the pregnant women in eastern Ethiopia had iron deficiency. Improving dietary diversity, meal frequency, and prenatal follow-up is essential to improve the high burden of ID and the adverse effect on pregnant women and the fetus. Moreover, a prospective study comparing maternal and perinatal outcomes among these spectra—iron depletion, ID, and IDA—is crucial for understanding their impact on maternal and perinatal mortality and morbidity.