Rhein (RH) has many bioactivities, but the application was limited of its poor solubility. The present study aimed to establish an efficient method for the synthesis of rhein amide derivatives (RAD) to increase the solubility and anti-tumour activity. RAD exhibited stronger anti-tumour activity than RH in MTT assay. The solubility and oil/water partition coefficient results indicated that rhein-phenylalanine and rhein-isoleucine have better absorption effect, which was consolidated in pharmacokinetic study. Then, rhein-phenylalanine and rhein-isoleucine were prepared into nanocrystals via the precipitation high-pressure homogenisation method. Additionally, the nanocrystals both displayed much higher dissolution profiles than the bulk drugs. Pharmacokinetics study indicated that the AUC(0-infinity) and C-max of nanocrystals increased markedly (p < 0.01). However, the concentration of RH-Phe-NC was far less than RH-lle-NC in plasma. Consequently, RH-He-NC was validated to be an applicable way to improve the bioavailability of RH, which owns a promising future in clinical application.