We report a green approach to synthesize lactoperoxidase (LPO) enzyme and metal ions hybrid nanoflowers (HNFs) and investigate mechanism underlying formation and enhanced catalytic activity and stability under different experimental parameters. The HNFs formed of LPO enzyme purified from bovine milk and copper ions (Cu2+) were synthesized at two different temperatures (+4 degrees C and 20 degrees C) in PBS (pH 7.4). The effects of experimental conditions, pH and storage temperatures, on the activity and stability of LPO-copper phosphate HNFs were evaluated using guaiacol as a substrate in the presence of hydrogen peroxide (H2O2). Optimum pHs were determined as pH 8 and pH 6 for LPO-copper phosphate HNF and free LPO, respectively. LPO-copper phosphate HNF has higher activity than free LPO at each pHs. Activities of LPO-copper phosphate HNF at pH 6 and pH 8 were calculated as 70.48 EU/mg, 107.23 EU/mg, respectively while free LPO shows 45.78 EU/mg and 30.12 EU/mg, respectively. Compared with free LPO, LPO-copper phosphate HNFs exhibited similar to 160% and similar to 360% increase in activities at pH 6 and pH 8, respectively. Additionally, LPO-copper phosphate HNFs displayed perfect reusability after six cycles. Finally, we demonstrated that LPO-copper phosphate HNFs can be utilized as a nanosensor for detection of dopamine and epinephrine. (C) 2015 Elsevier B.V. All rights reserved.