Bi-Pr-O composite nanoflakes were synthesized with the aid of polyvinyl pyrrolidone (PVP) as the surfactant. The characterization of Bi-Pr-O composite nanoflakes was performed by X-ray diffraction and electron microscopy. The prepared nanoflakes consisted of tetragonal Bi2O3, hexagonal Pr2O3, and hexagonal Bi0.4Pr0.6O1.5 with smooth surface. The size of whole nanoflakes is less than 1 μm and thickness is less than 100 nm. The formation of the Bi-Pr-O composite nanoflakes can be explained by a PVP-assisted growth mechanism. A systematic study of electrochemical catalysis of L-cysteine using a Bi-Pr-O-composite-nanoflake-modified electrode was carried out. A pair of quasi-reversible anodic and cathodic peaks are observed at −0.69 and +0.02 V, respectively, on the Bi-Pr-O-composite-nanoflake-modified electrode in 2 mM L-cysteine and 0.1 M KCl solution. The current response of the Bi-Pr-O-composite-nanoflake-modified electrode is proportional to the L-cysteine concentration in the range of 0.001—2 mM with a detection limit of 0.33 μM. In addition, the Bi-Pr-O composite nanoflakes display satisfactory reproducibility and stability for the detection of L-cysteine.