Antimony (Sb) is a toxic and potentially carcinogenic element in the environment. The toxicity of Sb(III) is ten times that of Sb(V). Therefore, on-site monitoring technique for dissolved Sb species is crucial for the study of Sb environmental processes. In this study, an automated, portable, and cost-effective system was developed for field simultaneous analysis of Sb(III) and Sb(III + V) in natural waters. The system comprised a portable atomic fluorescence spectrometer equipped with a built-in electrochemical H2 generator to reduce the consumption of acid/borohydride solution and make the atomizer more stable for on-site analysis. Flow injection technique was also used to achieve on-line pretreatment of water samples, including filtration, acidification, pre-reduction, and hydride generation procedures. Under the optimal conditions, the limits of detection (3σ, n = 11) of the developed method were 0.015 μg/L and the linear ranges were 0.05–5.0 μg/L for both Sb(III) and Sb(III + V). The relative standard deviations (n = 11) of the spiked samples of Sb(V) were 3.2% (0.05 μg/L), 3.3% (0.2 μg/L), and 1.7% (0.5 μg/L), respectively. The spiked recoveries of lake water, treated wastewater, and seawater ranged from 97.0% to 108.5%. The novel system of flow injection coupled with hydride generation atomic fluorescence spectrometer (FI-HG-AFS) was applied to carry out an 18-h fixed-point monitoring at a secondary settling tank of a wastewater treatment facility in Xiamen University, and a 6-h real-time underway analysis in the surface seawater of Dongshan Bay, China, proving that the system was capable of long-term monitoring in the field.