Background: In human body, oxidative stress plays an important role in generation of free radicals which intern contributes in generation of many disease conditions. Plant extracts are rich in various antioxidants which are having free radical scavenging activity. The aim of present study was to estimate the phytochemical constituents, to explore high performance thin layer chromatography (HPTLC) fingerprinting, to determine antioxidant potential, and to evaluate anti-anemic effect of an Amaranthus blitum L. leaf aqueous extract. Results: An aqueous extract of Amaranthus blitum L. leaves revealed the presence of flavonoids, glycosides, phenolics, tannins and triterpenoids. The extract was found to have DPPH (53.78 ± 0.66), ABTS (38.53 ± 5.7) and Superoxide scavenging activity (65.73 ± 0.9) and the results were similar and statistically significant as like standard antioxidant ascorbic acid (1000 μg/ml). The total phenolic content of the extract was 1696.06μg gallic acid equivalent/g; and the total flavonoid content was 1529.88±10.38μg quercetin equivalent/g. The HPTLC results revealed multi-colored bands of the varying intensities, which confirmed the presence of the polyphenols and flavonoids like active constituents in this extract. Treatment with Phenylhydrazine decreased Red blood cell count, Hemoglobin content and Hematocrit %. However, administration of this aqueous extract reversed the hematological changes induced by phenyl hydrazine. Conclusion: This study confirmed that, Amaranthus blitum L. leaves aqueous extract possess multidirectional, antioxidant and free radicals scavenging activity which is because of presence of the polyphenolic and flavonoid content into it. These findings provide evidence that the aqueous leaf extract of Amaranthus blitum L is a potential source of natural antioxidants and free radicals scavenging compounds. Also antioxidant properties of Amaranthus blitum L. leaves aqueous extract have positive impact on inhibition of hemolytic anemia induced by phenylhydrazine.