To achieve the prefabrication of concrete open-web sandwich slabs, a prefabricated concrete open-web sandwich slab structure with UHPC connections is proposed. Taking the prefabricated concrete open-web beam with UHPC connections as the research object, one cast-in-place concrete open-web beam specimen and one prefabricated concrete open-web beam specimen with UHPC connections were designed and fabricated. Firstly, hammer impact tests were conducted under simply supported conditions at both ends to analyze the first four natural frequencies, damping ratios, and mode shapes of each specimen. Subsequently, four-point bending loading tests were carried out to investigate their failure modes, load-deflection responses, and reinforcement strain evolution patterns. The results show that the failure process of the prefabricated concrete open-web beam is similar to that of the cast-in-place concrete open-web beam, both exhibiting ductile flexural failure. The yield load and peak load of the prefabricated concrete open-web beam are 67.63 kN and 76.93 kN, respectively, which are 3.8% and 4.3% higher than those of the cast-in-place concrete open-web beam, with a ductility coefficient of 5.26, indicating better flexural performance than the cast-in-place concrete open-web beam. Furthermore, the mode shapes and the first two natural frequencies of the prefabricated concrete open-web beam are essentially consistent with those of the cast-in-place concrete open-web beam, demonstrating comparable dynamic performance.