Oncolytic adenoviruses represent a promising therapeutic medicine for human cancer therapy, but successful translation to human clinical trials requires careful evaluation of these viral characteristics. While the function of the adenovirus proteins have been analyzed in detail, the dynamics of adenovirus infection remain largely unknown due to technological constraints which prevent adequate tracking of the adenovirus particles after infection. Fluorescent labeling of the adenoviral particles is one new strategy designed to directly analyze dynamic processes of viral infection in virus-host cell interactions. We hypothesized that the double labeling technique of adenovirus with fluorescent proteins would allow us to properly analyze intracellular viruses and the fate of viral proteins in live analysis of adenovirus as compared to a single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor capsid protein IX (pIX-mRFP1) and a green fluorescent minor core protein V (pV-EGFP), resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, the growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed approximately 150-fold reduced production of the viral progeny at 48 hours post-infection (h.p.i.) as compared to Ad5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and the nucleolus, respectively, at 18 h.p.i. These proteins were observed in the nucleus during the late stage of infection and the relocalization of the proteins was observed in an adenoviral replication-dependent manner. These results indicate that the simultaneous detection of adenovirus using dual-fluorescent proteins is suitable for real-time analysis, including identification of infected cells, and monitoring viral spread, which will be required for complete evaluation of oncolytic adenoviruses.