Scientific collaboration has become an important part of the people-to-people exchanges in the Belt and Road initiative, and remarkable progress has been made since 2013. Taking the 65 countries along the Belt and Road (BRI countries) as the research areas and using collaborated Web of Science (WOS) core collection papers to construct an international scientific collaboration matrix, the paper explores the spatial structure, hierarchy and formation mechanisms of scientific collaboration networks of 65 countries along the Belt and Road. The results show that: 1) Beyond the Belt and Road regions (BRI regions), Central & Eastern Europe, China and West Asia & North Africa have formed a situation in which they all have the most external links with other countries beyond BRI regions. China has the dominant role over other BRI countries in generating scientific links. The overall spatial structure has changed to a skeleton structure consisting of many dense regions, such as Europe, North America, East Asia and Oceania. 2) Within the Belt and Road regions, Central & Eastern Europe has become the largest collaboration partner with other sub-regions in BRI countries. The spatial structure of scientific collaboration networks has transformed from the ‘dual core’ composed of China and the Central & Eastern Europe region, to the ‘multi-polarization’ composed of ‘one zone and multi-points’. 3) The hierarchical structure of scientific collaboration networks presents a typical ‘core-periphery’ structure, and changes from ‘single core’ to ‘double cores’. 4) Among the formation mechanisms of scientific collaboration networks, scientific research strength and social proximity play the most important roles, while geographical distance gradually weakens the hindrance to scientific collaboration.