The upper reaches of the Yangtze River is one of the most abundant biodiversity areas in China, and also one of the global biodiversity hotspots. To obtain precise biodiversity information is a foundation of scientific decision-making for biodiversity conservation. The broad-scale biodiversity assessment has already become an urgent problem in the study and administration of biodiversity. This paper created a comprehensive index system and calculating expressions for evaluating regional biodiversity that were composed of vegetation landscape diversity index, nature reserve index, species diversity index based on various ecosystem types, and national protected species (flora and fauna) diversity index. We use this method to evaluate the importance of biodiversity conservation in the upper reaches of the Yangtze River by the county area as the basic assessment unit. The results of the assessment show that: (1) there are 18 counties with the richest biodiversity accounting for 4.95% of total county number; (2) 41 counties with richer biodiversity accounting for 1126%; (3) 76 counties with rich biodiversity accounting for 20.88%; (4) 106 counties with moderate biodiversity accounting for 29.12%; (5) 68 counties with below moderate biodiversity accounting for 18.68%; and (6) 55 counties with poor biodiversity accounting for 15.11%. All counties belonging to above-mentioned rich biodiversity mainly distribute in the Hengduan Mountains, Qinling-Daba Mountains, rainy zone of west China, and boundary mountains across Sichuan province, Chongqing city, Hubei province and Guizhou province.