As an important class of natural products, pyrazole alkaloids have a large number of applications in various aspects such as medicines and pesticides. Due to the special position of its amino group, 3-aminopyrazole is easy to form a hydrogen bond donor-acceptor-donor, and the hydrogen bond “zipper” structure enhances the binding ability of such compounds to receptors. This work provides a new one-pot three-components reaction for the efficient construction of multi-substituted aminopyrazoles derivatives via iodine-mediated cyclization in the presence of ethanol. After that, the potential mechanism of the reaction was explored based on the control transformation experimental and density functional theory (DFT) calculations. In addition, all obtained multi-substituted aminopyrazole derivatives were fully investigated for their application as potential anti-cancer agents, and some of which have been proved to exhibit excellent anti-cancer activity against A875, HepG2, and HL-7702 cell lines, and the possible structure-activity relationships also has been discussed based on the screening results. The kinase assay and molecular docking experimental indicate that this class of compounds may be a potential CDK1 inhibitors.