The shellfish has been extensively applied as a proxy for marine ecosystem monitoring, and the climate evolution or the shell growth might be derived from the elementary spatial distribution when using calcium (Ca) as the internal reference. Differed from other technique, the calcium emission lines are spread all over the wavelength range when applying Micro-probe Laser Induced Breakdown Spectroscopy (Micro-LIBS) to analyze the shell. So it is crucial to select a suitable calcium line for the shell elementary analysis. In this paper, the scallop shell composition was analyzed by comparing calcium internal references of total-line, atomic line (Ca Ⅰ 422.7 nm/Ca Ⅰ 487.9 nm) and ionic line (Ca Ⅱ 393.3 nm). It was found that, the fluctuation of Sr/Ca ratio was less than 7% and 10% in the investigated area when using total-line and strongest atomic line (Ca Ⅰ 422.7 nm) as the reference respectively, which was in agreement with the shell growth development. What’s more, the obtained results indicated that the element ratio Li/Ca had a high correlation to K/Ca and Na/Ca rather than Mn/Ca, so the light elements might be accumulated in a same pattern during the shell growth development. It was suggested that Micro-LIBS was a practical way for the seashell investigation, and a better elementary analysis could be achieved by using the internal reference of calcium total-line or the strong atomic line, while not in case of calcium ionic line.