The present study is focused on bioavailability and antioxidant property of carotenoids from microalgal biomass elucidated in rat model. Microalgal biomass containing 200 μM equivalent of β-carotene, astaxanthin and lutein per rat from Spirulina platensis, Haematococcus pluvialis and Botryococcus braunii biomass respectively was dispersed in olive oil and administered to rats for a period of 15 days. The levels of these carotenoids in the plasma, liver and eye were examined by high performance liquid chromatography and also confirmed by mass spectroscopy. The maximum peak levels (nmol/g) of β-carotene (615.61 ± 85.54), astaxanthin (896.51 ± 101.76) and lutein (679.55 ± 74.08) were detected in the liver followed by the eye and plasma. Astaxanthin accumulation in rats fed with H. pluvialis group was higher when compared to S. platensis and B. braunii groups. In H. pluvialis fed group, antioxidant enzymes catalase, superoxide dismutase, peroxidase and lipid peroxidation levels were high in the plasma and liver compared to S. platensis and B. braunii fed groups. These results indicate that the astaxanthin from H. pluvialis has better bioavailability and better antioxidant properties compared to other carotenoids.