Abstract: In order to determine the bioactivity of eukaryotes astaxanthin synthase ectopically expressed in tobacco, and to reveal the molecular basis for the development of tobacco bioreactor for astaxanthin, we codon-optimized and synthesized three eukaryotic-type astaxanthin synthases from Adonis aestivalis, Haematococcus pluvialis and Xanthophyllomyces dendrorhous respectively, and compared their astaxanthin synthetic bioactivities in tobacco using both transient and stable expression experiments. The results showed that the genes of the three resources were all successfully expressed in tobacco, whereas only the A. aestivalisand and H. pluvialis-derived synthases could specifically synthesize astaxanthin, and the A. aestivalis-derived astaxanthin synthases possessed a higher activity. The results also suggested that the stable expression system was more sensitive than the transient expression system in determining the activity of astaxanthin synthetic enzymes in tobacco. This study has provided molecular basis for the construction of tobacco bioreactor for astaxanthin synthesis.