Moss (as a reference material) and camphor (Cinnamomum Camphora) leaf, branch bark and bark samples were systematically collected across an urban-rural gradient in Guiyang (SW China) to determine the efficacy of using these bio-indicators to evaluate nitrogen (N) pollution. The tissue N concentrations (0.13%–2.70%) and δ¹⁵N values (−7.5‰ to +9.3‰) of all of these bio-indicators exhibited large spatial variations, as they recorded higher values in urban areas that quickly decreased with distance from the city center; moreover, both soil N concentrations and soil δ¹⁵N values were found no significant differences within each 6 km from the urban to the rural area. This not only suggests that the different N uptake strategies and variety of N responses of these bio-indicators can be reflected by their different susceptibilities to variations in N deposition but also reveals that they are able to indicate that urban N deposition is mostly from traffic and industry (NO_x-N), whereas rural N deposition is mainly from agriculture (NH_x-N). Compared to previously collected urban moss and camphor leaf samples, the significantly increased δ¹⁵N values in current urban moss and camphor leaf samples further indicate a greater contribution of NO_x-N than NH_x-N to urban N deposition. The feasibility of using the N concentrations and δ¹⁵N values of branch bark and bark as biomarkers of N deposition thus was further confirmed through the comparative use of these bio-indicators. It can be concluded that vascular plant leaves, branch bark and bark can be used as useful biomonitoring tools for evaluating atmospheric N pollution. For further study, quantitative criteria for the practical use of these bio-indicators in response to N deposition should be developed and the differences in the δ¹⁵N values of different plant parts should also be considered, particularly in urban environments that are severely disrupted by atmospheric pollution.