The aim of the present work is to apply a non-invasive test, using thumb fingerprint residue analysis, for detection of beta-thalassemia (β-Thal). The relative percentages of free amino acids (AA) in the latent fingerprint of β-Thal patients and healthy subjects were compared. The sample included 24β-Thal patient and 24 healthy subjects, aged 5-10 years old. Twenty-three AA plus ammonia were analyzed by a sensitive high-performance liquid chromatographic method with fluorescence detection. The profile of AA was calculated based on the percentage of relative amount of each AA to serine (Ser) as it found to be the predominant peak. The statistical and chromatographic profiles of β-Thal patients were characterized by significant decrease of ornithine, lysine, and zero tyrosine, with significant increase of ammonia, and proline. Other amino acids that exist in low ratios were estimated statistically for significance changes. The relative percentages of each AA of healthy subjects were approximately constant. For this reason, these mentioned AA were assigned as major fingerprint markers of β-Thal.