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An approach employing ultrafast laser hybrid subtractive-additive microfabrication, which combines ablation, three-dimensional nanolithography, and welding, is proposed for the realization of a lab-on-chip (LOC) device. A single amplified Yb:KGW femtosecond (fs)-pulsed laser source is shown to be suitable for fabricating microgrooves in glass slabs, polymerization of fine-meshes microfilter out of hybrid organic–inorganic photopolymer SZ2080 inside them, and, finally, sealing the whole chip with cover glass into a single monolithic piece. The created microfluidic device proved its particle sorting function by separating 1- and 10-μm polystyrene spheres in an aqueous mixture. All together, this proves that laser microfabrication based on a single amplified fs laser source is a flexible and versatile approach for the hybrid subtractive-additive manufacturing of functional mesoscale multimaterial LOC devices. [ABSTRACT FROM AUTHOR]