Regulatory intron insertion into cDNA can greatly enhance nuclear transgene expression in many eukaryotic hosts, especially in the green microalga Chlamydomonas reinhardtii where intron spreading is required to enable reliable and robust expression. We determined here that the first intron of C. reinhardtii rbcS2 (rbcS2i1) improves nuclear transgene expression 5.5-fold over eight other endogenous introns. For the convenient, reliable and standardized design of intron-containing transgenes, we developed the web tool “Intronserter”: https://bibiserv.cebitec.uni-bielefeld.de/intronserter. Intronserter implements back translation/codon optimization of input amino acid sequence(s) with user-defined codon usage tables, removes undesired sequence elements, performs systematic intron insertion to minimize exon lengths, and generates ready-to-synthesize DNA sequences for expression of any target. Although by default Intronserter will optimize transgenes for algal nuclear expression, all parameters are fully customizable. This flexibility enables its application to any other target organism and will encourage the broader implementation of advanced transgene design strategies in other eukaryotic hosts.