To achieve an efficient separation and purification of α-L-rhamnosidase (Rha) from naringinase (Nar) that was prepared from a fermented broth of Aspergillus niger, improved experimental methods were developed with aid of a chemical dithiothreitol (DTT) and a novel HPLC method. The addition of DTT did not negatively affect the purification of Rha and Nar, but greatly simplified the purification steps due to its strong capability of separating the Rha from Nar. The novel HPLC method enabled simultaneous measurement and differentiation of the Rha and the Nar with the naringin as the substrate. These improvements resulted in an efficient purification of the homogenous Rha with an estimated molecular weight of 87 kDa. Otherwise, Rha could not be extracted with enough purity even by the combination of sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The modified methods have ensured efficient purification of the Rha from A. niger naringinase. This study provides a powerful and simple procedure to separate and purify the Rha of Nar, which will facilitate further more in-depth studies of these enzymes, as well as their industrial applications.