Background: The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. Results: By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained similar to 150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also constructed, which increased the efficiency of gene interruption. Conclusion: In summary, this library provides a multifunctional platform both for obtaining mutants of interested genes and for screening of mutants with specific phenotype.