The electrodeless biocompatible manipulation of femtoliter‐scale aqueous microdroplets remains challenging to date. The appropriate isolation of electrostatic charges from fL‐scale aqueous microdroplets is crucial for electrodeless optoelectronic manipulation based on space‐charge‐density modulation. Here, we propose surfactant‐mediated photovoltaic manipulation, in which the surfactant layers self‐assembled at the water‐oil and oil‐LN interfaces are employed to isolate photovoltaic charges. The reduced electrostatic attenuation, remarkable hydrophobicity, and strong electrical breakdown suppression of the surfactant layers enable the stable and swift manipulation of fL‐scale aqueous microdroplets using μW‐level laser in an oil medium. By virtue of the surfactant‐mediated photovoltaic manipulation, we realized a controllable merging/touching/detaching switch of aqueous microdroplets by adjusting the laser illumination intensity and position and demonstrated the cascading biochemical operations and microreactions of aqueous microdroplets and microdroplet strings. To demonstrate its potential in photonic MEMS assemblies, we demonstrated the end coupling of a tightly focused‐laser‐beam into a ZnO microrod leveraging the refraction effect occurring at the water/oil interface. Moreover, because of the selective permeability of the droplet‐interface‐bilayer developed between the touching microdroplets, we achieved in situ adjustment of the size of the microdroplets and the fluorescent solute contained in the microdroplets, aiming at constructing multicomponent fluorescent microdroplets with tunable whispering‐gallery‐mode (WGM) characteristics.This article is protected by copyright. All rights reserved