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We very recently reported a novel aptamer biosensing concept based on a dual recognition mechanism originating from the small target-induced formation of a functional nucleic acid assembly. This assembly is constituted of a hairpin aptamer (named aptaswitch) for which the apical loop of the parent aptamer is substituted by a short RNA sequence prone to loop-loop interactions. It can switch between folded and unfolded states in the presence and in the absence of targets, respectively. The apical loop of the folded aptaswitch is then recognized by a second hairpin (called aptakiss), forming a kissing complex that signals the presence of the target. In the present work, we focus on the design improvement of this biosensing platform by using a previously described adenosine-adenoswitch couple as a model system and a fluorophore-labeled aptakiss as a reporting probe for fluorescence anisotropy (FA) detection. In the first step, the initially described adenoswitch was re-engineered to optimally convert the unfolded structure into the active stem-loop form upon adenosine binding. To further improve the assay performance, a blocking DNA oligonucleotide of the adenoswitch sequence was subsequently introduced into the assay scheme. This blocking strategy led to a significant increase in the FA response by reducing the background signal generated by the undesired binding of the free adenoswitch to the aptakiss probe. We obtained a detection limit which is fivefold lower than that observed with the previously reported kissing complex-based sensor. Finally, the optimized biosensing platform was successfully applied under biologically relevant conditions, i.e., diluted human serum, suggesting the potential practical applicability of the kissing sensing approach.