Acetylcholine (ACh) is a key cholinergic neurotransmitter that regulates synaptic signaling and neural function. Abnormal ACh levels have been linked to several neurodegenerative conditions, which makes fast and sensitive ACh quantification valuable for both research and diagnostics. Here, we developed a miniaturized, all-fiber microfluidic fluorescence sensor for ACh detection using pH-responsive cadmium telluride quantum dots (QDs) as the fluorescent probe. The sensing probe was implemented by splicing an exposed-core fiber (ECF) segment between multimode fibers and packaging the assembly in a glass capillary microchannel, enabling robust, alignment-free fluorescence collection and fiber-coupled readout. The proposed detection strategy was based on acetylcholinesterase-catalyzed hydrolysis of ACh, which generated acetic acid (HAc) and decreased local pH, leading to fluorescence quenching of the CdTe QDs. Under optimized conditions, the sensor exhibited a good linear response to ACh over the concentration range of 200–800 μM, with an estimated limit of detection of 13.2 μM. The readout response time was approximately 5 s, while the enzymatic hydrolysis was completed off- chip prior to readout. The device required only microliter-level sample volume and supported online fluorescence acquisition using a compact, fiber-coupled readout configuration. This work provides a rapid, stable, and selective ACh sensing approach that combines quantum-dot fluorescence with fiber-integrated microfluidics, offering a practical route toward miniaturized biochemical analysis and point-of-need testing.