Modified nucleic acids have a wide range of applications in many areas of biochemistry. In particular, fluorescence-based nucleic acid systems have been studied extensively for their implementation in molecular biology as platforms for disease diagnosis. In this Account, we summarize the past two decades of our research efforts associated with fluorescent nucleic acid systems for bio-sensing. We discuss single-nucleotide polymorphism–based molecular systems and their fluorescence behaviors in duplex DNA; the effects of fluorophore labeling on the fluorescence signals of modified oligonucleotides; pH-responsive nucleic acid–modified fluorescence biosensors; the use of double-stranded DNA and three-way junction–based molecular beacons (MBs) for the detection of biologically important mRNA and miRNA; quencher-free MBs and their applications in vivo; biologically significant G-quadruplex structures and i-motif–based fluorescent biosensors; the detection of trinucleotide repeats; and, finally, some other applications of modified nucleosides with unique properties. Fluorescence-based nucleic acid systems have been studied extensively for their implementation in molecular biology as platforms for disease diagnosis. In this Account, we discuss the study of single-nucleotide polymorphisms, duplex DNA, pH-dependent oligodeoxynucleotides (ODNs), i-motifs, G-quadruplexes, trinucleotide repeats, quencher-free molecular beacons, and three-way junction–based ODN systems through fluorescence phenomena, and their applications in specific areas.