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The interaction between K(2)Cr(2)O(7) and urease was investigated using fluorescence, UV-vis absorption, and circular dichroism (CD) spectroscopy. The experimental results showed that the fluorescence quenching of urease by K(2)Cr(2)O(7) was a result of the formation of K(2)Cr(2)O(7)-urease complex. The apparent binding constant K (A) between K(2)Cr(2)O(7) and urease at 295, 302, and 309 K were obtained to be 2.14 × 10(4), 1.96 × 10(4), and 1.92 × 10(4) L mol(-1), respectively. The thermodynamic parameters, ΔH° and ΔS° were estimated to be -5.90 kJ mol(-1), 43.67 J mol(-1) K(-1) according to the Van't Hoff equation. The electrostatic interaction played a major role in stabilizing the complex. The distance r between donor (urease) and acceptor (K(2)Cr(2)O(7)) was 5.08 nm. The effect of K(2)Cr(2)O(7) on the conformation of urease was analyzed using UV-vis absorption, CD, synchronous fluorescence spectroscopy, and three-dimensional fluorescence spectra, the environment around Trp and Tyr residues were altered.