In this work, reverse Atom Transfer Radical Polymerization (ATRP) was used for synthesising novel water-insoluble lineal copolymers which were used for designing new fluorescent and pH-sensitive, nanostructured films based on nanofibre mats made by electrospinning. The copolymers are based on the copolymerization of fluorescein o-acrylate (fluorescent pH-sensitive monomer) with methyl methacrylate and hydroxyl ethyl methacrylate (principal framework of the copolymers) and 3-methacryloylaminopropyl-trimethylammonium chloride (positively charged monomer) or 2-acrylamido-2-methylpropane sulfonic acid (negatively charged monomer). The electrostatic properties of these copolymers were investigated, and perfectly adjusted to maintain the complete insolubility of these materials in aqueous media. The copolymers were also characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H-NMR) spectroscopy and by triple detection gel permeation chromatography (GPC). The synthesised copolymers are soluble in organic solvents such as tetrahydrofuran, acetone, and dimethylformamide. These facts make them highly versatile and very good candidates for the preparation of novel nanostructured, optical pH-sensitive sensing phases produced by different techniques such as electrospinning, spin coating, deep coating, spray dry, spray coating, etc., and with different structures, morphologies, and geometries for many different applications. To demonstrate these claims, we used these copolymers for providing highly fluorescent pH-sensitive nanofibres made by electrospinning. The selected flow rates and voltages of the electrospinning configuration allowed the collection of dry fibres in nonwoven mats. The nanofibres were characterised by scanning electron microscopy (SEM) and fluorescence microscopy and the fibre mats were successfully used for pH-monitoring in two different pH ranges (6.5–8 and 8–10) showing reversibility, high sensitive and low response times.