Since the first reports dating back to the mid-1990s, ensembles and arrays of nanoelectrodes (NEEs and NEAs, respectively) have gained an important role as advanced electroanalytical tools thank to their unique characteristics which include, among others, dramatically improved signal/noise ratios, enhanced mass transport and suitability for extreme miniaturization. From the year 2000 onward, these properties have been exploited to develop electrochemical biosensors in which the surfaces of NEEs/NEAs have been functionalized with biorecognition layers using immobilization modes able to take the maximum advantage from the special morphology and composite nature of their surface. This paper presents an updated overview of this field. It consists of two parts. In the first, we discuss nanofabrication methods and the principles of functioning of NEEs/NEAs, focusing, in particular, on those features which are important for the development of highly sensitive and miniaturized biosensors. In the second part, we review literature references dealing the bioanalytical and biosensing applications of sensors based on biofunctionalized arrays/ensembles of nanoelectrodes, focusing our attention on the most recent advances, published in the last five years. The goal of this review is both to furnish fundamental knowledge to researchers starting their activity in this field and provide critical information on recent achievements which can stimulate new ideas for future developments to experienced scientists.