Several different approaches have been taken to development of homogeneous fluorescent aptamer assays including end-labeled beacons and signaling aptamers which are intrinsically quenched by nucleotides. Two new strategies dubbed “intrachain” and “competitive” FRET-aptamer assays are summarized in this review. Intrachain and competitive FRET-aptamers can be engineered on the molecular level through a series of exploratory experiments involving prior knowledge of aptamer secondary or tertiary structures and hypotheses about aptamer conformational changes. However, there is an intrinsic risk of altering aptamer affinity or specificity associated with chemical modifications of an aptamer. Natural selection methods for FRET-aptamers have also been devised to potentially obviate the chemical modification problem. The naturally selected aptamers are subjected to fluorophore (F)- and or quencher (Q)-conjugated nucleotide triphosphate (NTP) incorporation by polymerase chain reaction (PCR) with permissive polymerases such as Deep Vent exo-, but still demonstrate sensitive and specific assay performance despite modified bases, because they are ultimately selected after decoration with F and Q. This paper summarizes work in this area and presents some new examples of engineered and naturally selected FRET-aptamers for detection of vitamin D.