RNA-cleaving deoxyribozyme 8-17 has been increasingly used in nanotechnology and biosensing applications. Conventional methods to equip 8-17 with fluorescent signaling property usually involve covalent attachment of two dyes at nucleotide positions that are far away from the catalytic core, such that the bulky dye structures would not affect the deoxyribozyme activity. However, the maximum fluorescent enhancement associated with these 8-17 constructs is typically < or =10-fold, due to a high fluorescent background. To find an optimal balance between signal enhancement and signaling speed, we have conducted a comprehensive study on the effects of the nature of dyes (Alexa Fluor 488, 546 and 647; QSY 9 and 21) as well as their attaching positions along the substrate strand on the catalytic and signaling performance of 8-17. Our results have indicated that 8-17 is able to cleave almost every modified substrate, including those that have chromophores only 1 nt away from the cleavage site. Most importantly, almost all of these substrates are able to generate 15- to 85-fold signal enhancement within 10 min. We have also provided guidelines for selecting substrates that could offer the best signal enhancement, the fastest signaling speed, or the best balance between signal enhancement and signaling speed.