Acetaldehyde, a prevalent carbonyl compound in fermented foods, poses challenges in various applications due to its reactivity. This study addresses the need for efficient acetaldehyde detection methods across biotechnological, environmental, pharmaceutical, and food sectors. Herein, we present a novel colorimetric/UV spectrophotometric approach utilizing gold nanoparticles (AuNPs), particularly gold nano-flowers (AuNFs), for sensitive acetaldehyde identification. The method exhibits a notable sensitivity, detecting acetaldehyde at concentrations as low as 0.1 μM. The mechanism involves the interaction of acetaldehyde molecules with AuNFs, leading to a significant change in the absorbance spectrum, which serves as the basis for detection. Moreover, its applicability extends to human biofluids, notably urine samples. Integration with a cost-effective one-drop microfluidic colorimetric device (OD-μPCD) enables the development of an affordable test strip (CATS). This semi-analytical device, employing a multichannel OD-μPCD, facilitates real-time analysis of acetaldehyde in human samples. Our findings demonstrate the pioneering utilization of AuNPs for selective and sensitive acetaldehyde detection, promising advancements in environmental and occupational safety standards, and laying a foundation for enhanced detection and monitoring of related volatile organic compounds (VOCs).