Roads, as the main artificial linear structures in any landscape, have an important role in the vulnerability of natural ecosystems and their sustainability. The purpose of this research is to introduce a systematic method for ecological vulnerability assessment to be used in road site selection and environmental impact assessment procedure. Five steps of the vulnerability assessment are involved in this study; like determining the indices of vulnerability dimensions, calculating and mapping the indices, standardization of the indices, determining the vulnerability index and analysis of local variability. Lorestan province was selected as the case study due to its ecological properties and the presence of important road network to transfer goods and passengers. Indices like infrastructure fragmentation index, fractal dimension, residential neighborhood index, road traffic noise, erosion, topographic position index, and landscape connectivity index and dominance degree have been applied to quantify sensitivity, exposure and adaptive capacity as major components of the vulnerability. The findings show that the highest tension of road events is on the woodland habitat in 157270 ha. Besides, the regions with a high degree of sensitivity cover 28/1% (795132 ha) of the total study area. Wetland and temperate grassland habitats have the least adaptive capacity. Furthermore, the vulnerability classes in very low, low, moderate, high, and very high cover 2/5%, 52%, 42/1%, 3/1% and 0/3% of the total study area respectively. Therefore, approximately half of the province has the vulnerability degree in over average. This study showed the importance of the ecological vulnerability evaluation in environmental impact studies of development projects.