Objective To explore a rapid, convenient and non-polluting method of collecting cerebrospinal fluid (CSF) in Sprague Dawley (SD) rats for beginners. Methods Forty-five SD rats were divided into three groups — group 1 (collecting CSF through neck muscles), group 2 (collecting CSF through skin directly), and group 3 (collecting CSF through endorhachis). Results The successful rate of the CSF collection in the group 1 was 86.7%, group 2 was 46.7%, and group 3 is 33.3%. Conclusions The modified collection method of CSF by cisterna puncture through neck muscles in SD rats is simple, convenient, efficient and non-polluting, and is especially suitable for beginners.