The synthesis of copper nanoclusters (CuNCs) and its application as optical probe have attracted great attention. In this work, it was found that bovine serum albumin (BSA) reacted with copper ion (Cu2+) in a base medium to form a stable BSA-Cu complex, and the introduction of hydrogen peroxide (H2O2) could remarkably accelerate the formation of CuNCs. At the same time, the fluorescence intensity increased rapidly. Based on the fluorescence “on” response of BSA-Cu system, a kinetics method was developed for the fluorescent detection of H2O2. The fluorescence intensity of BSA-Cu linearly increased with the increase of H2O2 concentration in the range from 1.0 × 10−6 M to 1.5 × 10−3 M with the detection limit of 3.1 × 10−7 M (S/N = 3). After that, the collected BSA-Cu solution was placed until its fluorescence intensity reached the maximal value, during which the Cu2+ ions were fully changed into CuNCs. The experiment results demonstrated that the addition of L-cysteine (L-cys) into the solution led to an obvious fluorescence quenching. Based on the fluorescence “off” response of BSA-Cu system, an analytical method was established for the fluorescent determination of L-cys. The fluorescence intensity linearly reduced with the increase of L-cys concentration in the range of 2.0 × 10−4–1.0 × 10−2 M with the detection limit of 5.7 × 10−5 M (S/N = 3). Finally, the resulted BSA-Cu waste was treated by high temperature ashing and then dissolved with sulfuric acid, thus the CuNCs were turned into Cu2+ ions. The obtained Cu2+ solution continued to be used for the detection of H2O2 and L-cys in the next cycle. In this work, the cycle detection of H2O2 and L-cys and reuse of copper could be achieved through the conversion between Cu2+ and CuNCs. This method, with characteristics of high sensitivity, low cost and environment-friendliness, can be widely used for routine analysis of H2O2 and L-cys.