Calcium imaging has been widely used to monitor the activity of various neurons in C. elegans. However, it is a challenge to determine the calcium transient in a freely moving worm for two reasons. One reason is the challenge in ensuring the co-expression of the genetically encoded calcium indicator and reference fluorescent protein in the same target neurons. Another reason is the common problem with spectrum cross-talk between the fluorescent pair used most often: G-CaMPs (calcium indicator) and DsReds (the reference fluorescent protein). Spectrum cross-talk occasionally introduces artifacts in the calcium transient measurements. Herein, we developed a novel bicistronic expression strategy to ensure co-expression efficiency and simplify the labeling for the target neurons. Moreover, we presented a new fluorescent protein pair for calcium imaging, mKate2 and G-CaMPs, which have less spectrum cross-talk. We successfully tested our new labeling strategy in the C. elegans ASH sensory neuron. We anticipate that this improved technique will facilitate neural circuit studies in C. elegans.