The Pseuodomonads are well recognized for their ability to rapidly evolve σ⁵⁴-transcriptional activators to detect synthetic compounds in their environment, many of which are of high interest for replacement using microbial production processes.� Here we present a facile strategy for the in vivo detection and quantification of structurally-diverse, industrially-important metabolites on the single cell level.� We constructed an in vivo biosensor responding to C2-C8 linear alcohols from a rationally-designed library of chimeric Pseudomonad σ⁵⁴-transcriptional activators.� We focused on a putative alcohol-responsive transcription factor from Pseudomonas butanovora, BmoR, and XylR, a well characterized toluene-responsive transcription factor from Pseudomonas putida.� When transformed into Escherichia coli the biosensor yielded a linear response to exogenously added n-butanol up to 0.5% v/v; above which butanol-induced growth inhibition was observed.� In butanol production strains of E. coli the biosensor demonstrated accurate quantification of butanol titers as compared to gas chromatography-mass spectrometry measurements.� We then employed the biosensor for directed evolution of Escherichia coli for improved n-butanol production, targeting modifications to the E. coli genome and a heterologous n-butanol pathway from Clostridium acetobutylicum.� This work demonstrates a versatile strategy for rapid design of high-throughput screens and selections targeting for intracellular metabolites.� Further, we gained increased insight into possible mechanisms through which the nature is able to evolve the ability to detect and respond to synthetic compounds in the environment.