The design, synthesis and biological evaluation of a series of isoquinoline-based hydroxamic acid compounds as novel HDACs inhibitors were reported herein. A detailed SAR study showed most of the compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6. The IC50 values of compound 10 c against HDAC1, 3, 6 were 4.17 ± 0.11 nM, 4.00 ± 0.10 nM, 3.77 ± 0.07 nM, respectively. Most of the compounds showed great anti-proliferative activities against RPMI 8226, HCT 116 and Hep G2 cells. The IC50 values of compounds 10 a-h against RPMI 8226 cancer cell proliferation were all below 1 μM. HCT 116 cell was sensitive to the compounds 10 a, 10 f-g and 18 a with the IC50 values <0.3 μM. The active compounds 10a-d did not show inhibitory activity against hERG channel. All these evidence indicated these compounds had great potential as HDACs inhibitors for the further development.