This study focused on the design and synthesis of two Cu-based coordination polymers, [Cu2(4-dpye)(5-HSIP)(μ3-O)(H2O)2]·3H2O (Cu-CP-1) and [Cu(4-dpye)0.5(BCA)2] (Cu-CP-2), where 4-dpye = N,N'-bis(4-pyridinecarboxamide)-1,2-ethane, 5-H3SIP = 5-sulfoisophthalic acid, and HBCA = benzoic acid, by using a hydrothermal method. Single-crystal X-ray diffraction (SCXRD) study revealed that by adding various auxiliary ligands, the architectures of the Cu-CPs could be altered, yielding two distinct multinuclear Cu clusters. Moreover, the Cu-CPs can be used as urease inhibitors (UIs). In vitro experiments showed that the Cu-CPs had good urease inhibition effects with IC50 values of 0.53 ± 0.01 μM for Cu-CP-1 and 1.44 ± 0.01 μM for Cu-CP-2 and 98.48% (Cu-CP-1) and 96.27% (Cu-CP-2) inhibition of urease was achieved at a concentration of 100 μM, respectively. Furthermore, the inhibition effect of the tetranuclear Cu-CP was better than that of the binuclear Cu-CP. To better understand the potential mechanism of inhibition of the two copper complexes, we performed kinetic analysis using Lineweaver-Burk (L-B) plots in the presence of different concentrations of urea and different concentrations of inhibitors, and both Cu-CP-1 and Cu-CP-2 showed a non-competitive mode of inhibition. In addition, molecular docking analysis showed that the Cu-CPs were able to enter well into the urease binding pocket, thus interacting with key amino acid residues of urease to different degrees. Both kinetic and molecular docking studies theoretically explain and demonstrate the inhibition effect of both Cu-CPs on urease activity in vitro, which is expected to provide reasonable guidance and effective strategies for the development of novel, efficient, stable and safe CP-based UIs.