BACKGROUND: The traditional analytical methods for β-thalassemia carriers are time-consuming and costly. This study takes advantage of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to directly analyze human whole blood samples and established a preliminary method for screening β-thalassemia carriers. METHODS: A sample of 3 µL whole blood was diluted 200-fold with distilled water, mixed with sinapic acid, and subjected to MALDI-TOF MS analysis. The efficacy of the method for the screening of β-thalassemia carriers was assessed using the β-thalassemia gene results as the standard. RESULTS: β-thalassemia gene results showed 109 β-thalassemia carriers and 110 normal individuals out of 219 samples. We developed a δ-globin peak slope index that can simply and effectively distinguish β-thalassemia carriers from healthy individuals, the best cutoff value for the slope index was -0.819. CONCLUSIONS: MALDI-TOF MS has the potential to be a valuable tool for β-thalassemia carriers screening.